Use of neuropilin-1 (nrp1) as a cell surface marker for isolating human cardiac ventricular progenitor cells

ABSTRACT

The present invention provides NRP1 as a cell surface marker for isolating human cardiomyogenic ventricular progenitor cells (HVPs), in particular progenitor cells that preferentially differentiate into cardiac ventricular muscle cells. Additional HVP cell surface markers identified by single cell sequencing are also provided. The invention provides in vitro methods of the separation of NRP1+ ventricular progenitor cells, and the large scale expansion and propagation thereof. Large clonal populations of isolated NRP1+ ventricular progenitor cells are also provided. Methods of in vivo use of NRP1+ ventricular progenitor cells for cardiac repair or to improve cardiac function are also provided. Methods of using the NRP1+ ventricular progenitor cells for cardiac toxicity screening of test compounds are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to, and the benefit of, U.S.Provisional Application No. 62/549,345, filed on Aug. 23, 2017, theentire contents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Heart failure, predominantly caused by myocardial infarction, is theleading cause of death in both adults and children worldwide and isincreasing exponentially worldwide (Bui, A. L. et al. (2011) Nat. Rev.Cardiol. 8:30-41). The disease is primarily driven by the loss ofventricular muscle that occurs during myocardial injury (Lin, Z. and Pu,W. T. (2014) Sci. Transl. Med. 6:239rv1) and is compounded by thenegligible ability of the adult heart to mount a regenerative response(Bergmann, O. et al. (2009) Science 324:98-102; Senyo, S. E. et al.(2013) Nature 493:433-436). Although heart transplantation can becurative, the markedly limited availability of human heart organ donorshas led to a widespread unmet clinical need for a renewable source ofpure, mature and functional human ventricular muscle tissue (Segers, V.F. M. and Lee, R. J. (2008) Nature 451:937-942; Später, D. et al. (2014)Development 141:4418-4431).

Human pluripotent stem cells (hPSCs) offer the potential to generatelarge numbers of functional cardiomyocytes for potential clinicalrestoration of function in damaged or diseased hearts. Transplantationof stem cells into the heart to improve cardiac function and/or toenrich and regenerate damaged myocardium has been proposed (see e.g.,U.S. Patent Publication 20040180043). Combination therapy, in whichadult stem cells are administered in combination with treatment withgrowth factor proteins has also been proposed (see e.g., U.S. PatentPublication 20050214260).

While cell transplantation into the heart offers a promising approachfor improving cardiac function and regenerating heart tissue, thequestion of what type(s) of cells to transplant has been the subject ofmuch investigation. Types of cells investigated for use in regeneratingcardiac tissue include bone marrow cells (see e.g., Orlic, D. et al.(2001) Nature 410:701-705; Stamm, C. et al. (2003) Lancet 361:45-46;Perin, E. C. et al. (2003) Circulation 107:2294-2302), adult stem cells(see e.g., Jackson, K. A. et al. (2001) J. Clin. Invest. 107:1395-1402),bone marrow-derived mesenchymal stem cells (see e.g., Barbash, I. M. etal. (2003) Circulation 108:863; Pettinger, M. F. and Martin, B. J.(2003) Circ. Res. 95:9-20), bone marrow stromal cells (Bittira, B. etal. (2003) Eur. J. Cardiothorac. Surg. 24:393-398), a combination ofmesenchymal stem cells and fetal cardiomyocytes (see e.g., Min, J. Y. etal. (2002) Ann. Thorac. Surg. 74:1568-1575) and a combination of bonemarrow-derived mononuclear cells and bone marrow-derived mesenchymalstem cells (see e.g., U.S. Patent Publication 20080038229).Dedifferentiation of adult mammalian cardiomyocytes in vitro to generatecardiac stem cells for transplantation has also been proposed (see e.g.,U.S. Patent Publication 20100093089).

A significant advancement in the approach of cell transplantation toimprove cardiac function and regenerate heart tissue was theidentification and isolation of a family of multipotent cardiacprogenitor cells that are capable of giving rise to cardiac myocytes,cardiac smooth muscle and cardiac endothelial cells (Cai, C. L. et al.(2003) Dev. Cell. 5:877-889; Moretti, A. et al. (2006) Cell127:1151-1165; Bu, L. et al. (2009) Nature 460:113-117; U.S. PatentPublication 20060246446). These cardiac progenitor cells arecharacterized by the expression of the LIM homeodomain transcriptionfactor Islet 1 (Isl1) and thus are referred to as Isl1+ cardiacprogenitor cells. (Ibid). In contrast, Isl1 is not expressed indifferentiated cardiac cells. Additional markers of the Isl1+ cardiacprogenitor cells that arise later in differentiation than Isl1 have beendescribed and include Nkx2.5 and flk1(see e.g., U.S. Patent Publication20100166714).

The renewal and differentiation of Isl1+ cardiac progenitor cells hasbeen shown to be regulated by a Wnt/beta-catenin signaling pathway (seee.g., Qyang, Y. et al. (2007) Cell Stem Cell. 1:165-179; Kwon, C. et al.(2007) Proc. Natl. Acad. Sci. USA 104:10894-10899). This has led to thedevelopment of methods to induce a pluripotent stem cell to enter theIsl1+ lineage and for expansion of the Isl1+ population throughmodulation of Wnt signaling (see e.g., Lian, X. et al. (2012) Proc.Natl. Acad. Sci. USA 109:E1848-57; Lian, X. et al. (2013) Nat. Protoc.8:162-175; U.S. Patent Publication 20110033430; U.S. Patent Publication20130189785).

While human pluripotent stem cells hold great promise, a significantchallenge has been the ability to move from simply differentiation ofdiverse cardiac cells to forming a larger scale pure 3D ventricularmuscle tissue in vivo, which ultimately requires vascularization,assembly and alignment of an extracellular matrix, and maturation.Toward that end, a diverse population of cardiac cells (atrial,ventricular, pacemaker) has been coupled with artificial anddecellurized matrices (Masumoto, H. et al. (2014) Sci. Rep. 4:5716; Ott,H. C. et al. (2008) Nat. Med. 14:213-221; Schaaf, S. et al. (2011) PLoSOne 6:e26397), vascular cells and conduits (Tulloch, N. L. et al. (2011)Circ. Res. 109:47-59) and cocktails of microRNAs (Gama-Garvalho, M. etal. (2014) Cells 3:996-1026) have been studies, but the goal remainselusive.

While the identification of Isl1 as a marker of cardiac progenitor cellswas a significant advance, since Isl1 is an intracellular protein it isnot a suitable marker for use in isolating large quantities of viablecells. Rather, a cell surface marker(s) is still needed. Furthermore,Isl1 as a marker identifies a population that can differentiate intomultiple cell types within the cardiac lineage, and thus there is stilla need for markers that identify cardiac progenitor cells that arebiased toward a particular cell type within the cardiac lineage, inparticular for progenitor cells that differentiate into ventricularcells. Accordingly, there is still a great need in the art foradditional markers of cardiac progenitor cells, in particularcell-surface markers of cardiac progenitor cells, that predominantlygive rise to cardiomyocytes and that would allow for rapid isolation andlarge scale expansion of cardiomyogenic progenitor cells. Furthermore,there is still a great need in the art for methods and compositions forisolating cardiac ventricular progenitors, which differentiate intoventricular muscle cells in vivo, thereby allowing for transplantationof ventricular progenitors or ventricular muscle cells in vivo toenhance cardiac function.

SUMMARY OF THE INVENTION

This invention describes the use of Neuropilin-1 (NRP1) as a cellsurface marker for isolating human cardiac ventricular progenitor cells.Furthermore, additional cell surface markers suitable for use inisolating human cardiac ventricular progenitor cells are provided, asshown in Tables 1, 5 and 10. These human cardiac progenitor cells arebiased toward the ventricular lineage such that they differentiatepredominantly into ventricular muscle cells both in vitro and in vivo.That is, these cardiac progenitor cells can be cultured under conditionsin vitro such that they are biased toward the ventricular lineage andthus are human ventricular progenitor (HVP) cells. Moreover, whenintroduced into the ventricular region of the heart in a subject, theseprogenitor cells differentiate almost exclusively into ventricularmuscle cells that function according to their ventricular programming.In particular, the human ventricular progenitor cells provided hereinutilize a cell autonomous pathway by which these cells can build a pure3-D vascularized, functional and mature ventricular cell wall in vivo onthe surface of normal murine kidney or heart, thereby allowing fororgan-on-organ in vivo tissue engineering.

Using single cell sequencing of cardiac progenitors at different stagesof differentiation, a panel of genes differentially expressed in HVPswas identified, as described in Example 14 and shown in Table 10. Withinthis panel, day 5 progenitor cells were shown to express both Islet 1(Isl1) and NRP1. This identification of a key cell surface marker ofcardiac ventricular progenitor cells allows for easy and rapid isolationof the cells. Furthermore, determination of culture conditions forexpansion and ventricular lineage bias of the cells allows for thepreparation of large cultures (a billion or more cells) of a clonalpopulation of cardiac ventricular progenitor cells. These cells can beused, for example, to improve function in a damaged heart in a subject,particularly damage in the ventricular region. The progenitor cells canbe transplanted in vivo for differentiation into ventricular cells insitu or, alternatively, a heart muscle patch, comprising ventricularmuscle cells, can be prepared in vitro from the progenitors forsubsequent transplantation in vivo. The cells also can be used, forexample, in in vitro toxicity screening assays to evaluate the cardiactoxicity of test compounds, as well as for biochemical studies toidentify relevant pathways used in cardiac maturation anddifferentiation.

Thus, the invention provides human cardiac ventricular progenitor cellsin purified form. The human cardiac ventricular progenitors are capableof differentiation into ventricular muscle cells in vitro and in vivo.These progenitor cells can be expanded to large numbers of cells invitro and when transplanted into the ventricular region of the heart invivo they differentiate essentially exclusively into ventricular musclecells. Still further, the cells have the capacity to migrate in vivo todifferent sites and, when transplanted in vivo the cells does what theyare programmed to do as a ventricular cell (as opposed to a cardiacmyocyte which simply contracts). Thus, the ventricular progenitor cellscan be grafted to native tissue to enhance ventricular function and havethe ability to call in vasculature into the new ventricular tissue.

Accordingly, in one aspect, the invention pertains to a method forisolating human cardiac ventricular progenitor cells, the methodcomprising:

contacting a culture of cells containing human cardiac progenitor cellswith one or more agents reactive with NRP1; and

separating NRP1 reactive positive cells from non-reactive cells tothereby isolate human cardiac ventricular progenitor cells.

In one embodiment, the human cardiac progenitor cells are contacted bothwith an agent reactive with NRP1 and with at least one second agent thatbinds to an HVP marker(s) (as described herein) to thereby separateNRP1/second agent reactive positive cells from non-reactive cells.

Preferably, the human cardiac progenitor cells are Islet 1+ humancardiac progenitor cells. Preferably, the human cardiac progenitor cellsare JAG1+, FZD4+, LIFR+, FGFR3+ and/or TNFSF9+. In another embodiment,the culture of cells is also contacted with at least one second agent;and NRP1 reactive/second agent reactive positive cells are separatedfrom non-reactive cells to thereby isolate cardiac ventricularprogenitor cells. In one embodiment, the at least one second agent bindsa marker selected from JAG1+, FZD4+, LIFR+, FGFR3+ and/or TNFSF9+. Theculture of cells can be simultaneously contacted with the agent reactivewith NRP1 and the at least one second agent. Alternatively, the cultureof cells can be contacted with the at least one second agent beforecontacting with the agent reactive with NRP1. Alternatively, the cultureof cells can be contacted with the agent reactive with NRP1 beforecontacting with the at least one second agent. In another embodiment,the culture of cells is also negatively selected for lack of expressionof at least one marker of pluripotent stem cells, such as TRA-1-60. Inanother embodiment, the human cardiac ventricular progenitor cells arefurther cultured and differentiated such that they express theventricular marker MLC2v. In certain embodiments, the starting cultureof cells containing human cardiac progenitor cells is derived from humanembryonic stem cells or human induced pluripotent cells.

In another aspect, the invention pertains to a method for isolatinghuman cardiac ventricular progenitor cells, the method comprising:

culturing human pluripotent stem cells under conditions that generatecardiac progenitor cells to obtain a cultured cell population;

contacting the cultured cell population with one or more agents reactivewith NRP1; and

separating NRP1 reactive positive cells from non-reactive cells tothereby isolate human cardiac ventricular progenitor cells.

In another embodiment, the culture of cells is also contacted with atleast one second agent; and NRP1 reactive/second agent reactive positivecells are separated from non-reactive cells to thereby isolate cardiacventricular progenitor cells. In one embodiment, the at least one secondagent binds a marker selected from JAG1+, FZD4+, LIFR+, FGFR3+ and/orTNFSF9+. The culture of cells can be simultaneously contacted with theagent reactive with NRP1 and the at least one second agent.Alternatively, the culture of cells can be contacted with the at leastone second agent before contacting with the agent reactive with NRP1.Alternatively, the culture of cells can be contacted with the agentreactive with NRP1 before contacting with the at least one second agent.In another embodiment, the culture of cells is also negatively selectedfor lack of expression of at least one marker of pluripotent stem cells,such as TRA-1-60. In another embodiment, the human cardiac ventricularprogenitor cells are further cultured and differentiated such that theyexpress the ventricular marker MLC2v. In certain embodiments, thestarting culture of cells containing human cardiac progenitor cells isderived from human embryonic stem cells or human induced pluripotentcells.

In the methods for isolating human cardiac ventricular progenitor cells,various types of agents that bind to NRP1 can be used as the agent(s)reactive with NRP1. For example, in one embodiment, the agent reactivewith NRP1 is an anti-NRP1 antibody, such as a monoclonal antibody. Inanother embodiment, the agent reactive with NRP1 is a soluble NRP1ligand, such as a NRP1 ligand fusion protein. For example, the agentreactive with NRP1 can comprise the NRP1 ligand VEGF-A or Sema3A, suchas a soluble fusion protein (e.g., an Ig fusion protein).

In the methods for isolating human cardiac ventricular progenitor cells,various types of separation methods can be used to separate NRP1reactive positive cells from non-reactive cells. For example, in oneembodiment, the reactive positive cells are separated from thenon-reactive cells by fluorescence activated cell sorting (FACS). Inanother embodiment, the reactive positive cells are separated from thenon-reactive cells by magnetic activated cells sorting (MACS).

In yet another aspect, the invention pertains to a method of obtaining aclonal population of human cardiac ventricular progenitor cells, themethod comprising:

isolating a single NRP1+ human cardiac ventricular progenitor cell; and

culturing the single NRP1+ human cardiac ventricular progenitor cellunder conditions such that the cell is expanded to at least 1×10⁹ cellsto thereby obtain a clonal population of human cardiac ventricularprogenitor cells.

In one embodiment, the single NRP1+ human cardiac ventricular progenitorcell is Islet 1 positive, Nkx2.5 negative and flk1 negative at the timeof initial culture. The single NRP1+ human cardiac ventricularprogenitor cell can be isolated by methods such as those described above(e.g., FACS or MACS). The single NRP1+ human cardiac ventricularprogenitor cell can be isolated using a reagent(s) reactive with NRP1,such as those described above (e.g., anti-NRP1 antibodies, soluble NRP1ligands, such as ligand fusion proteins). Upon further culture anddifferentiation, the clonal population of human cardiac ventricularprogenitor cells can express the ventricular marker MLCV2.

In a preferred embodiment, the single NRP1+ human cardiac ventricularprogenitor cell is cultured in vitro under conditions such that the cellis biased toward ventricular differentiation. For example, the singleNRP1+ human cardiac ventricular progenitor cell can be cultured inCardiac Progenitor Culture (CPC) medium (80% advanced DMEM/F12supplemented with 20% KnockOut Serum Replacement, 2.5 mM GlutaMax and100 μg/ml Vitamin C), which allows for differentiation of the cells intoventricular cells expressing the MLC2v ventricular marker. In variousembodiments, the single NRP1+ human cardiac ventricular progenitor cellis expanded to a clonal population of, for example, at least 1×10⁹cells, at least 2×10⁹ cells, at least 5×10⁹ cells or at least 10×10⁹cells.

Accordingly in another aspect, the invention pertains to a clonalpopulation of isolated NRP1+ human cardiac ventricular progenitor cells.In various embodiments, this clonal population comprises, for example,at least 1×10⁹ cells, at least 2×10⁹ cells, at least 5×10⁹ cells or atleast 10×10⁹ cells. In a preferred embodiment, this clonal populationcomprises at least 1×10⁹ NRP1+ human cardiac ventricular progenitorcells.

In yet another aspect, the invention pertains to a method of enhancingcardiac function in a subject using the NRP1+ human cardiac ventricularprogenitor cells described herein. For example, in one embodiment, theinvention provides a method of enhancing cardiac function in a subject,the method comprising administering a pharmaceutical compositioncomprising a clonal population NRP1+ human cardiac ventricularprogenitor cells, such as a clonal population of at least at least 1×10⁹cells, at least 2×10⁹ cells, at least 5×10⁹ cells or at least 10×10⁹cells. In one embodiment, the clonal population is administered directlyinto the heart of the subject. For example, the clonal population can beadministered directly into a ventricular region of the heart of thesubject. In one embodiment, the pharmaceutical composition administeredto the subject comprises the clonal population formulated onto a threedimensional matrix, such as a heart muscle patch comprising ventricularmuscle cells. The subject is one in need of enhancement of cardiacfunction, for example someone who has suffered a myocardial infarctionor someone who has a congenital heart disorder.

In yet another aspect, the invention pertains to a method for generatinghuman ventricular tissue comprising:

transplanting NRP1+ human cardiac ventricular progenitor cells into anorgan of a non-human animal; and

allowing the progenitor cells to grow in vivo such that humanventricular tissue is generated.

The non-human animal can be, for example, an immunodeficient mouse. Theorgan can be, for example, the kidney (e.g., the cells are transplantedunder the kidney capsule) or the heart. In one embodiment, the cells aretransplanted at a time when one, two, three, four or five of thefollowing cell marker patterns are present: (i) after peak of cardiacmesoderm formation; (ii) at time of peak Islet-1 expression; (iii)before peak of NKX2.5 expression; (iv) before peak expression ofdownstream genes MEF-2 and TBX-1; and (v) before expression ofdifferentiated contractile protein genes. In one embodiment, the cellsare transplanted between day 5 and day 7 (inclusive) of in vitro cultureof human pluripotent stem cells under conditions to generate humanventricular progenitor cells. In another embodiment, the cells aretransplanted on day 6 of in vitro culture of human pluripotent stemcells under conditions to generate human ventricular progenitor cells.The method can further include harvesting the human ventricular tissuegenerated in the non-human animal.

In still another aspect of the invention, the human cardiac ventricularprogenitor cells described herein can be used in screening assays toevaluate the cardiac toxicity of a test compound. Accordingly, theinvention provides a method of screening for cardiac toxicity of testcompound, the method comprising:

providing NRP1+ cardiac ventricular progenitor cells;

contacting the cells with the test compound; and

measuring toxicity of the test compound for the cells,

wherein toxicity of the test compound for the cells indicates cardiactoxicity of the test compound. The toxicity of the test compound for thecells can be measured, for example, by assessing cell viability or otherphysiological parameters of the cell.

Culturing methods for generating human ventricular progenitor cells arealso provided. For example, in one embodiment, the invention pertains toa method of generating human ventricular progenitors (HVPs) comprising:

-   -   culturing human pluripotent stems cells (hPSCs) in a medium        comprising CHIR98014 such that cells expressing cardiac        mesodermal markers are generated; and    -   culturing the cells expressing cardiac mesodermal markers in a        medium comprising Wnt-C59 such that HVPs are generated.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an exemplary culturing protocol forgenerating human Isl1+cardiomyogenic progenitor cells from humanpluripotent stem cells (hPSCs).

FIG. 2 shows the results of Western blot analysis of protein expressionduring cardiac differentiation of hPSCs, showing expression of Isl1,Nkx2.5 and cTn1. GAPDH was used as a control.

FIG. 3 shows the results of flow cytometry analysis of cardiomyogenicprogenitor cells, showing expression of Isl1 on cells at day 6 ofdifferentiation.

FIG. 4 shows the results of double staining flow cytometry analysis ofcardiomyogenic progenitor cells, showing coexpression of Isl1 and Jag1on cells at day 6 of differentiation.

FIG. 5 shows the results of Western blot analysis of protein expressionduring cardiac differentiation of hPSCs, showing expression of FZD4.GAPDH was used as a control.

FIG. 6 shows the results of double staining flow cytometry analysis ofcardiomyogenic progenitor cells, showing coexpression of Isl1 and FZD4on cells at day 5 of differentiation.

FIG. 7 is a schematic diagram of the generation of human ventricularprogenitor (HVP) cells, their ultimate differentiation into ventricularmyocytes, their antibody purification and their use in transplantation.

FIGS. 8A and 8B are schematic diagrams of the transplantation of HPVsinto the renal capsule (FIG. 8A) or intra-myocardially (FIG. 8B) fororgan-on-organ tissue engineering.

FIG. 9 shows the results of double staining flow cytometry analysis ofhuman ventricular progenitor (HVP) cells, showing coexpression of Isl1and LIFR on the cells.

FIGS. 10A and 10B show the results of flow cytometry analysis of theexpression of LIFR and FGFR3 on human ventricular progenitor cells (FIG.10B) as compared to undifferentiated embryonic stem (ES) cells (FIG.10A).

FIG. 11 is a tSNE plot of day 5 cells from single cell sequencing,showing two clusters of cells based on differential gene expression,labeled 0 and 1.

FIGS. 12A and 12B are tSNE plots of Isl1 expression (FIG. 12A) and NRP1expression (FIG. 12B) on day 5 cells from single cell sequencing. Darkgrey denotes high expression, middle grey denotes low expression andlight grey denotes no expression.

FIG. 13 is a violin plot of expression levels of Isl1 and NRP1 inclusters of day 5 cells from single cell sequencing, showing that NRP1+and ISL1+ cells are in the same cluster, since they have a similar geneexpression profile.

FIGS. 14A, 14B and 14C show the results of flow cytometric analysis ofthe expression of NRP1 (FIG. 14A), TRA-1-60 (FIG. 14B) and both NRP1 andTRA-1-60 (FIG. 14C) on day 6 HVPs generated from H9 stem cells.

FIGS. 15A, 15B and 15C show the results of flow cytometric analysis ofthe expression of NRP1 (FIG. 15A), ISL1 (FIG. 15B) and both NRP1 andISL1 (FIG. 15C) on day 6 HVPs generated from H9 stem cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods of isolating human cardiomyogenicprogenitor cells, in particular cells that are biased to the ventricularlineage, as well as isolated clonal populations of such progenitorcells, based on the discovery that NRP1 is a cell surface marker forcardiac ventricular progenitor cells (HVPs). Additional suitable cellssurface markers for HVPs are shown in, for example, Tables 1, 5 and 10.In vitro and in vivo uses for these cardiac ventricular progenitor cellsare also provided.

HVPs have previously been shown to express cell surface markers such asJAG1, FZD4, LIFR, FGFR3 and/or TNFSF9, as well as the intracellularmarker Islet 1 (see U.S. Ser. No. 14/832,324, filed Aug. 21, 2015, andU.S. Ser. No. 14/984,783, filed Dec. 30, 2015, the entire contents ofeach of which are expressly incorporated herein by reference).Furthermore, various positive and negative engraftment markers of HVPshave been identified (see U.S. Ser. No. 15/433,713, filed Feb. 15, 2017,the entire contents of which is expressly incorporated herein byreference).

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the terms “Neuropilin-1”, “NRP1” are usedinterchangeably to refer to a protein known in the art that has beendescribed in, for example, Soker, S. et al. (1998) Cell 92:735-745 andRossignol, M. et al. (2000) Genomics 70:211-222. NRP1 is also referredto in the art as BDCA4, VEGF165R and CD304. A non-limiting example of anNRP1 protein is the human protein having the amino acid sequence setforth in Genbank Accession Number NP_003864.4.

As used herein, the term “stem cells” is used in a broad sense andincludes traditional stem cells, progenitor cells, pre-progenitor cells,reserve cells, and the like. The term “stem cell” or “progenitor” areused interchangeably herein, and refer to an undifferentiated cell whichis capable of proliferation and giving rise to more progenitor cellshaving the ability to generate a large number of mother cells that canin turn give rise to differentiated, or differentiable daughter cells.The daughter cells themselves can be induced to proliferate and produceprogeny that subsequently differentiate into one or more mature celltypes, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers then, to a cellwith the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one embodiment, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell may derive from amultipotent cell which itself is derived from a multipotent cell, and soon. While each of these multipotent cells may be considered stem cells,the range of cell types each can give rise to may vary considerably.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. In manybiological instances, stem cells are also “multipotent” because they canproduce progeny of more than one distinct cell type, but this is notrequired for “stem-ness.” Self-renewal is the other classical part ofthe stem cell definition, and it is essential as used in this document.In theory, self-renewal can occur by either of two major mechanisms.Stem cells may divide asymmetrically, with one daughter retaining thestem state and the other daughter expressing some distinct otherspecific function and phenotype. Alternatively, some of the stem cellsin a population can divide symmetrically into two stems, thusmaintaining some stem cells in the population as a whole, while othercells in the population give rise to differentiated progeny only.Formally, it is possible that cells that begin as stem cells mightproceed toward a differentiated phenotype, but then “reverse” andre-express the stem cell phenotype, a term often referred to as“dedifferentiation”.

The term “progenitor cell” is used herein to refer to cells that have acellular phenotype that is more primitive (e.g., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells also have significant or veryhigh proliferative potential. Progenitor cells can give rise to multipledistinct differentiated cell types or to a single differentiated celltype, depending on the developmental pathway and on the environment inwhich the cells develop and differentiate.

The term “pluripotent” as used herein refers to a cell with thecapacity, under different conditions, to differentiate to cell typescharacteristic of all three germ cell layers (endoderm, mesoderm andectoderm). Pluripotent cells are characterized primarily by theirability to differentiate to all three germ layers, using, for example, anude mouse and teratomas formation assay. Pluripotency is also evidencedby the expression of embryonic stem (ES) cell markers, although thepreferred test for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. In someembodiments, a pluripotent cell is an undifferentiated cell.

The term “pluripotency” or a “pluripotent state” as used herein refersto a cell with the ability to differentiate into all three embryonicgerm layers: endoderm (gut tissue), mesoderm (including blood, muscle,and vessels), and ectoderm (such as skin and nerve), and typically hasthe potential to divide in vitro for a long period of time, e.g.,greater than one year or more than 30 passages.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and examples of multipotent cells include adult stem cells,such as for example, hematopoietic stem cells and neural stem cells.Multipotent means a stem cell may form many types of cells in a givenlineage, but not cells of other lineages. For example, a multipotentblood stem cell can form the many different types of blood cells (red,white, platelets, etc.), but it cannot form neurons.

The term “embryonic stem cell” or “ES cell” or “ESC” are usedinterchangeably herein and refer to the pluripotent stem cells of theinner cell mass of the embryonic blastocyst (see U.S. Pat. Nos.5,843,780, 6,200,806, which are incorporated herein by reference). Suchcells can similarly be obtained from the inner cell mass of blastocystsderived from somatic cell nuclear transfer (see, for example, U.S. Pat.Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein byreference). The distinguishing characteristics of an embryonic stem celldefine an embryonic stem cell phenotype. Accordingly, a cell has thephenotype of an embryonic stem cell if it possesses one or more of theunique characteristics of an embryonic stem cell such that that cell canbe distinguished from other cells. Exemplary distinguishing embryonicstem cell characteristics include, without limitation, gene expressionprofile, proliferative capacity, differentiation capacity, karyotype,responsiveness to particular culture conditions, and the like. In someembodiments, an ES cell can be obtained without destroying the embryo,for example, without destroying a human embryo.

The term “adult stem cell” or “ASC” is used to refer to any multipotentstem cell derived from non-embryonic tissue, including fetal, juvenile,and adult tissue. Stem cells have been isolated from a wide variety ofadult tissues including blood, bone marrow, brain, olfactory epithelium,skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stemcells can be characterized based on gene expression, factorresponsiveness, and morphology in culture. Exemplary adult stem cellsinclude neural stem cells, neural crest stem cells, mesenchymal stemcells, hematopoietic stem cells, and pancreatic stem cells. As indicatedabove, stem cells have been found resident in virtually every tissue.Accordingly, the present invention appreciates that stem cellpopulations can be isolated from virtually any animal tissue.

The term “human pluripotent stem cell” (abbreviated as hPSC), as usedherein, refers to a human cell that has the capacity to differentiateinto a variety of different cell types as discussed above regarding stemcells and pluripotency. Human pluripotent human stem cells include, forexample, induced pluripotent stem cells (iPSC) and human embryonic stemcells, such as ES cell lines.

The term “human cardiac progenitor cell”, as used herein, refers to ahuman progenitor cell that is committed to the cardiac lineage and thathas the capacity to differentiate into all three cardiac lineage cells(cardiac muscle cells, endothelial cells and smooth muscle cells). Aculture of human cardiac progenitor cells can be obtained by, forexample, culturing stem cells under conditions that bias the stem cellstoward differentiation to the cardiac lineage. In certain embodiments,the stem cells that are cultured to generate human cardiac progenitorcells are human embryonic stem cells or human induced pluripotent cells.In certain embodiments, c-kit+ adult progenitor cells are explicitlyexcluded for use in generating human cardiac progenitor cells.

The term “human cardiomyogenic progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat predominantly differentiates into cardiac muscle cells (i.e., morethan 50% of the differentiated cells, preferably more than 60%, 70%, 80%or 90% of the differentiated cells, derived from the progenitor cellsare cardiac muscle cells).

The term “cardiac ventricular progenitor cell”, as used herein, refersto a progenitor cell that is committed to the cardiac lineage and thatpredominantly differentiates into cardiac ventricular muscle cells(i.e., more than 50% of the differentiated cells, preferably more than60%, 70%, 80% or 90% of the differentiated cells, derived from theprogenitor cells are cardiac ventricular muscle cells). This type ofcell is also referred to herein as a human ventricular progenitor, orHVP, cell.

The term “cardiomyocyte” refers to a muscle cell of the heart (e.g. acardiac muscle cell). A cardiomyocyte will generally express on its cellsurface and/or in the cytoplasm one or more cardiac-specific marker.Suitable cardiomyocyte-specific markers include, but are not limited to,cardiac troponin I, cardiac troponin-C, tropomyosin, caveolin-3, GATA-4,myosin heavy chain, myosin light chain-2a, myosin light chain-2v,ryanodine receptor, and atrial natriuretic factor.

The term “derived from” used in the context of a cell derived fromanother cell means that a cell has stemmed (e.g. changed from orproduced by) a cell that is a different cell type. The term “derivedfrom” also refers to cells that have been differentiated from aprogenitor cell. The term “Isl1+ cardiac progenitor cell”, as usedherein, refers to a human progenitor cell that is committed to thecardiac lineage and that expresses Islet 1.

The term “Isl1+ NRP1+ cardiac progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat expresses both Islet 1 and NRP1.

With respect to cells in cell cultures or in cell populations, the term“substantially free of” means that the specified cell type of which thecell culture or cell population is free, is present in an amount of lessthan about 10%, less than about 9%, less than about 8%, less than about7%, less than about 6%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2% or less than about 1% of the totalnumber of cells present in the cell culture or cell population.

In the context of cell ontogeny, the adjective “differentiated”, or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellit is being compared with. Thus, stem cells can differentiate tolineage-restricted precursor cells (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as an cardiomyocyte precursor), and thento an end-stage differentiated cell, which plays a characteristic rolein a certain tissue type, and may or may not retain the capacity toproliferate further.

The term “differentiation” in the present context means the formation ofcells expressing markers known to be associated with cells that are morespecialized and closer to becoming terminally differentiated cellsincapable of further differentiation. The pathway along which cellsprogress from a less committed cell, to a cell that is increasinglycommitted to a particular cell type, and eventually to a terminallydifferentiated cell is referred to as progressive differentiation orprogressive commitment. Cell which are more specialized (e.g., havebegun to progress along a path of progressive differentiation) but notyet terminally differentiated are referred to as partiallydifferentiated. Differentiation is a developmental process whereby cellsassume a specialized phenotype, e.g., acquire one or morecharacteristics or functions distinct from other cell types. In somecases, the differentiated phenotype refers to a cell phenotype that isat the mature endpoint in some developmental pathway (a so calledterminally differentiated cell). In many, but not all tissues, theprocess of differentiation is coupled with exit from the cell cycle. Inthese cases, the terminally differentiated cells lose or greatlyrestrict their capacity to proliferate. However, we note that in thecontext of this specification, the terms “differentiation” or“differentiated” refer to cells that are more specialized in their fateor function than at a previous point in their development, and includesboth cells that are terminally differentiated and cells that, althoughnot terminally differentiated, are more specialized than at a previouspoint in their development. The development of a cell from anuncommitted cell (for example, a stem cell), to a cell with anincreasing degree of commitment to a particular differentiated celltype, and finally to a terminally differentiated cell is known asprogressive differentiation or progressive commitment. A cell that is“differentiated” relative to a progenitor cell has one or morephenotypic differences relative to that progenitor cell. Phenotypicdifferences include, but are not limited to morphologic differences anddifferences in gene expression and biological activity, including notonly the presence or absence of an expressed marker, but alsodifferences in the amount of a marker and differences in theco-expression patterns of a set of markers.

The term “differentiation” as used herein refers to the cellulardevelopment of a cell from a primitive stage towards a more mature (i.e.less primitive) cell.

As used herein, “proliferating” and “proliferation” refers to anincrease in the number of cells in a population (growth) by means ofcell division. Cell proliferation is generally understood to result fromthe coordinated activation of multiple signal transduction pathways inresponse to the environment, including growth factors and othermitogens. Cell proliferation may also be promoted by release from theactions of intra- or extracellular signals and mechanisms that block ornegatively affect cell proliferation.

The terms “renewal” or “self-renewal” or “proliferation” are usedinterchangeably herein, and refers to a process of a cell making morecopies of itself (e.g. duplication) of the cell. In some embodiments,cells are capable of renewal of themselves by dividing into the sameundifferentiated cells (e.g. progenitor cell type) over long periods,and/or many months to years. In some instances, proliferation refers tothe expansion of cells by the repeated division of single cells into twoidentical daughter cells.

The term “lineages” as used herein refers to a term to describe cellswith a common ancestry or cells with a common developmental fate, forexample cells that have a developmental fate to develop into ventricularcardiomyocytes.

The term “clonal population”, as used herein, refers to a population ofcells that is derived from the outgrowth of a single cell. That is, thecells within the clonal population are all progeny of a single cell thatwas used to seed the clonal population.

The term “media” as referred to herein is a medium for maintaining atissue or cell population, or culturing a cell population (e.g. “culturemedia”) containing nutrients that maintain cell viability and supportproliferation. The cell culture medium may contain any of the followingin an appropriate combination: salt(s), buffer(s), amino acids, glucoseor other sugar(s), antibiotics, serum or serum replacement, and othercomponents such as peptide growth factors, etc. Cell culture mediaordinarily used for particular cell types are known to those skilled inthe art.

The term “phenotype” refers to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

A “marker” as used herein describes the characteristics and/or phenotypeof a cell. Markers can be used for selection of cells comprisingcharacteristics of interest. Markers will vary with specific cells.Markers are characteristics, whether morphological, functional orbiochemical (enzymatic) characteristics particular to a cell type, ormolecules expressed by the cell type. Preferably, such markers areproteins, and more preferably, possess an epitope for antibodies orother binding molecules available in the art. However, a marker mayconsist of any molecule found in a cell including, but not limited to,proteins (peptides and polypeptides), lipids, polysaccharides, nucleicacids and steroids. Examples of morphological characteristics or traitsinclude, but are not limited to, shape, size, and nuclear to cytoplasmicratio. Examples of functional characteristics or traits include, but arenot limited to, the ability to adhere to particular substrates, abilityto incorporate or exclude particular dyes, ability to migrate underparticular conditions, and the ability to differentiate along particularlineages. Markers may be detected by any method available to one ofskill in the art.

The term “isolated cell” as used herein refers to a cell that has beenremoved from an organism in which it was originally found or adescendant of such a cell. Optionally the cell has been cultured invitro, e.g., in the presence of other cells. Optionally the cell islater introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells as used herein refers to a population of cells that has beenremoved and separated from a mixed or heterogeneous population of cells.In some embodiments, an isolated population is a substantially purepopulation of cells as compared to the heterogeneous population fromwhich the cells were isolated or enriched from.

The term “substantially pure”, with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,preferably at least about 85%, more preferably at least about 90%, andmost preferably at least about 95% pure, with respect to the cellsmaking up a total cell population.

The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example a human, to whom cardiac ventricularprogenitor cells as disclosed herein can be implanted into, for e.g.treatment, which in some embodiments encompasses prophylactic treatmentor for a disease model, with methods and compositions described herein,is or are provided. For treatment of disease states that are specificfor a specific animal such as a human subject, the term “subject” refersto that specific animal. The terms “non-human animals” and “non-humanmammals” are used interchangeably herein, and include mammals such asrats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-humanprimates. The term “subject” also encompasses any vertebrate includingbut not limited to mammals, reptiles, amphibians and fish. However,advantageously, the subject is a mammal such as a human, or othermammals such as a domesticated mammal, e.g. dog, cat, horse, and thelike, or production mammal, e.g. cow, sheep, pig, and the like are alsoencompassed in the term subject.

As used herein, the term “recipient” refers to a subject that willreceive a transplanted organ, tissue or cell.

The term “three-dimensional matrix” or “scaffold” or “matrices” as usedherein refers in the broad sense to a composition comprising abiocompatible matrix, scaffold, or the like. The three-dimensionalmatrix may be liquid, gel, semi-solid, or solid at 25° C. Thethree-dimensional matrix may be biodegradable or non-biodegradable. Insome embodiments, the three-dimensional matrix is biocompatible, orbioresorbable or bioreplacable. Exemplary three-dimensional matricesinclude polymers and hydrogels comprising collagen, fibrin, chitosan,MATRIGEL™, polyethylene glycol, dextrans including chemicallycrosslinkable or photocrosslinkable dextrans, processed tissue matrixsuch as submucosal tissue and the like. In certain embodiments, thethree-dimensional matrix comprises allogeneic components, autologouscomponents, or both allogeneic components and autologous components. Incertain embodiments, the three-dimensional matrix comprises synthetic orsemi-synthetic materials. In certain embodiments, the three-dimensionalmatrix comprises a framework or support, such as a fibrin-derivedscaffold.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably and refer to the placement ofcardiomyogenic progenitor cells and/or cardiomyocytes differentiated asdescribed herein into a subject by a method or route which results in atleast partial localization of the cells at a desired site. The cells canbe administered by any appropriate route that results in delivery to adesired location in the subject where at least a portion of the cellsremain viable. The period of viability of the cells after administrationto a subject can be as short as a few hours, e.g. twenty-four hours, toa few days, to as long as several years.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value. The term “substantially” or “predominantly” as usedherein means a proportion of at least about 60%, or preferably at leastabout 70% or at least about 80%, or at least about 90%, at least about95%, at least about 97% or at least about 99% or more, or any integerbetween 70% and 100%.

The term “disease” or “disorder” is used interchangeably herein, andrefers to any alternation in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, indisposition oraffection.

As used herein, the phrase “cardiovascular condition, disease ordisorder” is intended to include all disorders characterized byinsufficient, undesired or abnormal cardiac function, e.g. ischemicheart disease, hypertensive heart disease and pulmonary hypertensiveheart disease, valvular disease, congenital heart disease and anycondition which leads to congestive heart failure in a subject,particularly a human subject. Insufficient or abnormal cardiac functioncan be the result of disease, injury and/or aging. By way of background,a response to myocardial injury follows a well-defined path in whichsome cells die while others enter a state of hibernation where they arenot yet dead but are dysfunctional. This is followed by infiltration ofinflammatory cells, deposition of collagen as part of scarring, all ofwhich happen in parallel with in-growth of new blood vessels and adegree of continued cell death. As used herein, the term “ischemia”refers to any localized tissue ischemia due to reduction of the inflowof blood. The term “myocardial ischemia” refers to circulatorydisturbances caused by coronary atherosclerosis and/or inadequate oxygensupply to the myocardium. For example, an acute myocardial infarctionrepresents an irreversible ischemic insult to myocardial tissue. Thisinsult results in an occlusive (e.g., thrombotic or embolic) event inthe coronary circulation and produces an environment in which themyocardial metabolic demands exceed the supply of oxygen to themyocardial tissue.

As used herein, the term “treating” or “treatment” are usedinterchangeably herein and refers to reducing or decreasing oralleviating or halting at least one adverse effect or symptom of acardiovascular condition, disease or disorder, i.e., any disordercharacterized by insufficient or undesired cardiac function. Adverseeffects or symptoms of cardiac disorders are well-known in the art andinclude, but are not limited to, dyspnea, chest pain, palpitations,dizziness, syncope, edema, cyanosis, pallor, fatigue and death. In someembodiments, the term “treatment” as used herein refers to prophylactictreatment or preventative treatment to prevent the development of asymptom of a cardiovascular condition in a subject.

Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced as that term is defined herein. Alternatively, atreatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms or decrease of markers of the disease, but also a cessation orslowing of progress or worsening of a symptom that would be expected inabsence of treatment. Beneficial or desired clinical results include,but are not limited to, alleviation of one or more symptom(s),diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already diagnosedwith a cardiac condition, as well as those likely to develop a cardiaccondition due to genetic susceptibility or other factors such as weight,diet and health. In some embodiments, the term to treat also encompassespreventative measures and/or prophylactic treatment, which includesadministering a pharmaceutical composition as disclosed herein toprevent the onset of a disease or disorder.

A therapeutically significant reduction in a symptom is, e.g. at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100%, at least about 125%,at least about 150% or more in a measured parameter as compared to acontrol or non-treated subject. Measured or measurable parametersinclude clinically detectable markers of disease, for example, elevatedor depressed levels of a biological marker, as well as parametersrelated to a clinically accepted scale of symptoms or markers for adisease or disorder. It will be understood, that the total daily usageof the compositions and formulations as disclosed herein will be decidedby the attending physician within the scope of sound medical judgment.The exact amount required will vary depending on factors such as thetype of disease being treated.

With reference to the treatment of a cardiovascular condition or diseasein a subject, the term “therapeutically effective amount” refers to theamount that is safe and sufficient to prevent or delay the developmentor a cardiovascular disease or disorder. The amount can thus cure orcause the cardiovascular disease or disorder to go into remission, slowthe course of cardiovascular disease progression, slow or inhibit asymptom of a cardiovascular disease or disorder, slow or inhibit theestablishment of secondary symptoms of a cardiovascular disease ordisorder or inhibit the development of a secondary symptom of acardiovascular disease or disorder. The effective amount for thetreatment of the cardiovascular disease or disorder depends on the typeof cardiovascular disease to be treated, the severity of the symptoms,the subject being treated, the age and general condition of the subject,the mode of administration and so forth. Thus, it is not possible tospecify the exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation. The efficacy oftreatment can be judged by an ordinarily skilled practitioner, forexample, efficacy can be assessed in animal models of a cardiovasculardisease or disorder as discussed herein, for example treatment of arodent with acute myocardial infarction or ischemia-reperfusion injury,and any treatment or administration of the compositions or formulationsthat leads to a decrease of at least one symptom of the cardiovasculardisease or disorder as disclosed herein, for example, increased heartejection fraction, decreased rate of heart failure, decreased infarctsize, decreased associated morbidity (pulmonary edema, renal failure,arrhythmias) improved exercise tolerance or other quality of lifemeasures, and decreased mortality indicates effective treatment. Inembodiments where the compositions are used for the treatment of acardiovascular disease or disorder, the efficacy of the composition canbe judged using an experimental animal model of cardiovascular disease,e.g., animal models of ischemia-reperfusion injury (Headrick J P, Am JPhysiol Heart circ Physiol 285; H1797; 2003) and animal models acutemyocardial infarction. (Yang Z, Am J Physiol Heart Circ. Physiol282:H949:2002; Guo Y, J Mol Cell Cardiol 33; 825-830, 2001). When usingan experimental animal model, efficacy of treatment is evidenced when areduction in a symptom of the cardiovascular disease or disorder, forexample, a reduction in one or more symptom of dyspnea, chest pain,palpitations, dizziness, syncope, edema, cyanosis, pallor, fatigue andhigh blood pressure which occurs earlier in treated, versus untreatedanimals. By “earlier” is meant that a decrease, for example in the sizeof the tumor occurs at least 5% earlier, but preferably more, e.g., oneday earlier, two days earlier, 3 days earlier, or more.

As used herein, the term “treating” when used in reference to atreatment of a cardiovascular disease or disorder is used to refer tothe reduction of a symptom and/or a biochemical marker of acardiovascular disease or disorder, for example a reduction in at leastone biochemical marker of a cardiovascular disease by at least about 10%would be considered an effective treatment. Examples of such biochemicalmarkers of cardiovascular disease include a reduction of, for example,creatine phosphokinase (CPK), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) in the blood, and/or a decrease in a symptom ofcardiovascular disease and/or an improvement in blood flow and cardiacfunction as determined by someone of ordinary skill in the art asmeasured by electrocardiogram (ECG or EKG), or echocardiogram (heartultrasound), Doppler ultrasound and nuclear medicine imaging. Areduction in a symptom of a cardiovascular disease by at least about 10%would also be considered effective treatment by the methods as disclosedherein. As alternative examples, a reduction in a symptom ofcardiovascular disease, for example a reduction of at least one of thefollowing; dyspnea, chest pain, palpitations, dizziness, syncope, edema,cyanosis etc. by at least about 10% or a cessation of such systems, or areduction in the size one such symptom of a cardiovascular disease by atleast about 10% would also be considered as affective treatments by themethods as disclosed herein. In some embodiments, it is preferred, butnot required that the therapeutic agent actually eliminate thecardiovascular disease or disorder, rather just reduce a symptom to amanageable extent.

Subjects amenable to treatment by the methods as disclosed herein can beidentified by any method to diagnose myocardial infarction (commonlyreferred to as a heart attack) commonly known by persons of ordinaryskill in the art are amenable to treatment using the methods asdisclosed herein, and such diagnostic methods include, for example butare not limited to; (i) blood tests to detect levels of creatinephosphokinase (CPK), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) and other enzymes released during myocardialinfarction; (ii) electrocardiogram (ECG or EKG) which is a graphicrecordation of cardiac activity, either on paper or a computer monitor.An ECG can be beneficial in detecting disease and/or damage; (iii)echocardiogram (heart ultrasound) used to investigate congenital heartdisease and assessing abnormalities of the heart wall, includingfunctional abnormalities of the heart wall, valves and blood vessels;(iv) Doppler ultrasound can be used to measure blood flow across a heartvalve; (v) nuclear medicine imaging (also referred to as radionuclidescanning in the art) allows visualization of the anatomy and function ofan organ, and can be used to detect coronary artery disease, myocardialinfarction, valve disease, heart transplant rejection, check theeffectiveness of bypass surgery, or to select patients for angioplastyor coronary bypass graft.

The terms “coronary artery disease” and “acute coronary syndrome” asused interchangeably herein, and refer to myocardial infarction refer toa cardiovascular condition, disease or disorder, include all disorderscharacterized by insufficient, undesired or abnormal cardiac function,e.g. ischemic heart disease, hypertensive heart disease and pulmonaryhypertensive heart disease, valvular disease, congenital heart diseaseand any condition which leads to congestive heart failure in a subject,particularly a human subject. Insufficient or abnormal cardiac functioncan be the result of disease, injury and/or aging. By way of background,a response to myocardial injury follows a well-defined path in whichsome cells die while others enter a state of hibernation where they arenot yet dead but are dysfunctional. This is followed by infiltration ofinflammatory cells, deposition of collagen as part of scarring, all ofwhich happen in parallel with in-growth of new blood vessels and adegree of continued cell death.

As used herein, the term “ischemia” refers to any localized tissueischemia due to reduction of the inflow of blood. The term “myocardialischemia” refers to circulatory disturbances caused by coronaryatherosclerosis and/or inadequate oxygen supply to the myocardium. Forexample, an acute myocardial infarction represents an irreversibleischemic insult to myocardial tissue. This insult results in anocclusive (e.g., thrombotic or embolic) event in the coronarycirculation and produces an environment in which the myocardialmetabolic demands exceed the supply of oxygen to the myocardial tissue.

The terms “composition” or “pharmaceutical composition” usedinterchangeably herein refer to compositions or formulations thatusually comprise an excipient, such as a pharmaceutically acceptablecarrier that is conventional in the art and that is suitable foradministration to mammals, and preferably humans or human cells. In someembodiments, pharmaceutical compositions can be specifically formulatedfor direct delivery to a target tissue or organ, for example, by directinjection or via catheter injection to a target tissue. In otherembodiments, compositions can be specifically formulated foradministration via one or more of a number of routes, including but notlimited to, oral, ocular parenteral, intravenous, intraarterial,subcutaneous, intranasal, sublingual, intraspinal,intracerebroventricular, and the like. In addition, compositions fortopical (e.g., oral mucosa, respiratory mucosa) and/or oraladministration can form solutions, suspensions, tablets, pills,capsules, sustained-release formulations, oral rinses, or powders, asknown in the art are described herein. The compositions also can includestabilizers and preservatives. For examples of carriers, stabilizers andadjuvants, University of the Sciences in Philadelphia (2005) Remington:The Science and Practice of Pharmacy with Facts and Comparisons, 21stEd.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably and refer to the placement of apharmaceutical composition comprising cardiomyogenic progenitor cells,or a composition comprising a population of differentiated cardiomyoctes(e.g., ventricular cardiomyocytes) as described herein, into a subjectby a method or route which results in at least partial localization ofthe pharmaceutical composition, at a desired site or tissue location. Insome embodiments, the pharmaceutical composition can be administered byany appropriate route which results in effective treatment in thesubject, i.e. administration results in delivery to a desired locationor tissue in the subject where at least a portion of the cells arelocated at a desired target tissue or target cell location.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration of cardiovascular stem cells and/or their progeny and/orcompound and/or other material other than directly into the cardiactissue, such that it enters the animal's system and, thus, is subject tometabolism and other like processes, for example, subcutaneous orintravenous administration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation.

The term “drug” or “compound” or “test compound” as used herein refersto a chemical entity or biological product, or combination of chemicalentities or biological products, administered to a subject to treat orprevent or control a disease or condition. The chemical entity orbiological product is preferably, but not necessarily a low molecularweight compound, but may also be a larger compound, for example, anoligomer of nucleic acids, amino acids, or carbohydrates includingwithout limitation proteins, oligonucleotides, ribozymes, DNAzymes,glycoproteins, siRNAs, lipoproteins, aptamers, and modifications andcombinations thereof.

The term “transplantation” as used herein refers to introduction of newcells (e.g. reprogrammed cells), tissues (such as differentiated cellsproduced from reprogrammed cells), or organs into a host (i.e.transplant recipient or transplant subject).

The term “agent reactive with NRP1”, as used herein, refers to an agentthat binds to or otherwise interacts with NRP1. Preferably, the agent“specifically” binds or otherwise interacts with NRP1 such that it doesnot bind or interact with other non-NRP1 proteins.

The term “antibody”, as used herein, includes whole antibodies and anyantigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers, in one preferred embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The term“antigen-binding portion” of an antibody (or simply “antibody portion”),as used herein, refers to one or more fragments of an antibody thatretain the ability to specifically bind to an antigen.

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a particularepitope.

The term “human monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which hasvariable and optional constant regions derived from human germlineimmunoglobulin sequences.

The term “humanized monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which has heavyand light chain CDR1, 2 and 3 from a non-human antibody (e.g., a mousemonoclonal antibody) grafted into human framework and constant regions.

The term “chimeric monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which has heavyand light chain variable regions from one species linked to constantregions from another species.

The term “fusion protein”, as used herein, refers to a compositeprotein, typically made using recombinant DNA technology, in which twodifferent proteins, or portions thereof, are operatively linkedtogether. A non-limiting example is an Fc fusion protein in which anon-immunoglobulin protein is operatively linked to an immunoglobulin Fcregion.

Various aspects of the invention are described in further detail in thefollowing subsections.

Methods of Isolating Human Cardiac Ventricular Progenitor Cells

In one aspect, the invention pertains to methods of isolating humancardiac ventricular progenitor cells. As described in the Examples, NRP1has now been identified as a cell surface marker of human cardiacventricular progenitor cells and thus this marker can be used tofacilitate isolation of these progenitor cells. Alternative to NRP1,additional markers for human cardiac ventricular progenitor cells areprovided herein, including the markers shown in Tables 1, 5 and 10. Anyof the proteins in these tables that is a cell surface protein can beused in the isolation of HVPs as described herein.

Accordingly, in one embodiment, the invention provides a method forisolating human cardiac ventricular progenitor cells, the methodcomprising:

contacting a culture of human cells containing cardiac progenitor cellswith one or more agents reactive with NRP1; and

separating NRP1 reactive positive cells from non-reactive cells tothereby isolate human cardiac ventricular progenitor cells.

Alternatively, after the contacting step, the method can compriseisolating NRP1 reactive positive cells from non-reactive cells tothereby isolate human cardiac ventricular progenitor cells.

Also as described in the Examples, Islet 1 is a marker that isco-expressed with NRP1 by the cardiac ventricular progenitor cells andthus both markers can be used to facilitate isolation of theseprogenitor cells. Accordingly, in another embodiment of the abovemethod, the culture of human cells is also contacted with an agentreactive with Islet 1; and NRP1 reactive/Islet 1 reactive positive cellsare separated from non-reactive cells to thereby isolate human cardiacventricular progenitor cells. Alternatively or additionally, agentsreactive with other HVP markers, including but not limited to JAG1,FZD4, LIFR, FGFR3 and/or TNFSF9, can be used in combination with NRP1 inthe isolation of HVPs. Accordingly, the culture of human cells can besimultaneously contacted with the agent(s) reactive with NRP1 and/or atleast one second agent reactive with an HVP marker(s), including but notlimited to JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9. In one embodiment, theculture of human cells is contacted with the agent reactive with NRP1before contacting with the second agent(s) reactive with an HVPmarker(s). In another embodiment, the culture of human cells iscontacted with the second agent(s) reactive with an HVP marker(s) beforecontacting with the agent reactive with NRP1.

In another embodiment, the method of isolating HVPs further comprisesnegatively selecting for at least one marker expressed on the surface ofhuman pluripotent stem cells, such as TRA-1-60. The use of negativeselection (in addition to positive selection for NRP1 expression, and/orexpression of other positive HVP markers) ensures a rigorous definitionof the HVP population as well as eliminating batch variation andpotential teratoma-causing cells. Accordingly, in one embodiment,cardiac progenitor cells are selected for lack of expression of at leastone marker expressed on the surface of human pluripotent stem cells(negative selection), such as TRA-1-60, to thereby isolate a highlypurified population of HVPs. Non-limiting examples of markers expressedon the surface of human pluripotent stem cells that can be used fornegative selection include: TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3,SSEA4, CD9, CD24, E-cadherin and Podocalyxin, and combinations thereof.

In another embodiment, the invention provides a method for isolatinghuman cardiac ventricular progenitor cells, the method comprising:

culturing human pluripotent stem cells under conditions that generatecardiac progenitor cells to obtain a culture of cells;

contacting the culture of cells with one or more agents reactive withNRP1; and separating NRP1 reactive positive cells from non-reactivecells to thereby isolate human cardiac ventricular progenitor cells.

Alternatively, after the culturing and contacting steps, the method cancomprise isolating NRP1 from non-reactive cells to thereby isolate humancardiac ventricular progenitor cells.

Alternatively or additionally, second agents reactive with other HVPmarkers, including but not limited to JAG1, FZD4, LIFR, FGFR3 and/orTNFSF9, can be used in combination with NRP1 in the isolation of HVPs.Accordingly, the culture of human cells can be simultaneously contactedwith the agent(s) reactive with NRP1 and/or at least one second agentreactive with an HVP marker(s), including but not limited to JAG1, FZD4,LIFR, FGFR3 and/or TNFSF9. In one embodiment, the culture of human cellsis contacted with the agent reactive with NRP1 before contacting withthe second agent(s) reactive with an HVP marker(s). In anotherembodiment, the culture of human cells is contacted with the secondagent(s) reactive with an HVP marker(s) before contacting with the agentreactive with NRP1.

In another embodiment, the method of isolating HVPs further comprisesnegatively selecting for at least one marker expressed on the surface ofhuman pluripotent stem cells, such as TRA-1-60. The use of negativeselection (in addition to positive selection for NRP1 expression, and/orexpression of other positive HVP markers) ensures a rigorous definitionof the HVP population as well as eliminating batch variation andpotential teratoma-causing cells. Accordingly, in one embodiment,cardiac progenitor cells are selected for lack of expression of at leastone marker expressed on the surface of human pluripotent stem cells(negative selection), such as TRA-1-60, to thereby isolate a highlypurified population of HVPs. Non-limiting examples of markers expressedon the surface of human pluripotent stem cells that can be used fornegative selection include: TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3,SSEA4, CD9, CD24, E-cadherin and Podocalyxin, and combinations thereof.

In a preferred embodiment, the agent reactive with NRP1 is ananti-NRP1antibody, such as a monoclonal antibody. Non-limiting examplesinclude murine, rabbit, human, humanized or chimeric monoclonalantibodies with binding specificity for NRP1. Anti-NRP1 monoclonalantibodies are commercially available in the art. Moreover, anti-NRP1antibodies can be prepared using standard techniques well established inthe art using NRP1 as the antigen.

In another embodiment, the agent reactive with NRP1 is a NRP1 ligand,such as a soluble NRP1 ligand or a soluble NRP1 ligand fusion protein.Non-limiting examples of NRP1 ligands include VEGF-A and Sema3A. SolubleNRP1 ligands can be prepared using standard recombinant DNA techniques,for example by deletion of the transmembrane and cytoplasmic domains. Asoluble ligand can be transformed into a soluble ligand fusion proteinalso using standard recombinant DNA techniques. A fusion protein can beprepared in which fusion partner can comprise a binding moiety thatfacilitates separation of the fusion protein.

In order to separate the NRP1 reactive positive cells from non-reactivecells, one of a variety of different cell separation techniques known inthe art can be used. Preferably, the NRP1 reactive positive cells areseparated from non-reactive cells by fluorescence activated cell sorting(FACS). The FACS technology, and apparatuses for carrying it out toseparate cells, is well established in the art. When FACS is used forcell separation, preferably the agent(s) reactive with NRP1 that is usedis a fluorescently-labeled anti-NRP1 monoclonal antibody. Alternatively,cell separation can be achieved by, for example, magnetic activated cellsorting (MACS). When MACS is used for cell separation, preferably theagent reactive with NRP1 that is used is magnetic nanoparticles coatedwith anti-NRP1 monoclonal antibody. Alternatively, other single cellsorting methodologies known in the art can be applied to the methods ofisolating cardiac ventricular progenitor cells of the invention,including but not limited to IsoRaft array and DEPArray technologies.

Prior to contact with the agent(s) reactive with NRP1, and separation ofNRP1 reactive cells, human pluripotent stem cells can be cultured underconditions that lead to the generation of cardiac progenitor cells.Culture conditions for generating cardiac progenitor cells have beendescribed in the art (see e.g., Lian, X. et al. (2012) Proc. Natl. Acad.Sci. USA 109:E1848-1857; U.S. Patent Publication No. 20130189785) andalso are described in detail in Example 1 and FIG. 1, as well as inExample 10. Typically, Wnt/β-catenin signaling is first activated in thehPSCs, followed by an incubation period, followed by inhibition ofWnt/β-catenin signaling. Activation of Wnt/β-catenin signaling isachieved by incubation with a Gsk3 inhibitor, preferably CHIR98014 (CAS556813-39-9). Inhibition of Wnt/β-catenin signaling is achieved byincubation with a Porcn inhibitor, preferably Wnt-C59 (CAS1243243-89-1). Suitable hPSCs for use in the methods of the inventioninclude induced pluripotent stem cells (iPSC), such as 19-11-1, 19-9-7or 6-9-9 cells (Yu, J. et al. (2009) Science 324:797-801), and humanembryonic stem cell lines, such as ES03 cells (WiCell ResearchInstitute) or H9 cells (Thomson, J. A. et al. (1998) Science282:1145-1147). Suitable culture media for generating cardiomyogenicprogenitors include E8 medium, mTeSR1 medium and RPMI/B27 minus insulin,each described further in Example 1 and/or Example 10.

Preferably, the human cardiomyogenic progenitor cells are ventricularprogenitor cells. Culture conditions have now been determined that biasthe cardiomyogenic progenitor cells to the ventricular lineage. Theseventricular cardiomyogenic progenitor cells can be cultured in RPMI/B27medium and they can further differentiate into ventricular muscle cells.A preferred medium for culturing the cardiac ventricular progenitorcells in vitro such that they differentiation into ventricular cells invitro (e.g., expressing the MLC2v marker described below) is the CardiacProgenitor Culture (CPC) medium (advanced DMEM/F12 supplemented with 20%KnockOut Serum Replacement, 2.5 mM GlutaMAX and 100 μg/ml Vitamin C).

Known markers of differentiated cardiac cells can be used to identifythe type(s) of cells that are generated by differentiation of thecardiac progenitor cells. For example, cardiac troponin I (cTnI) can beused as a marker of cardiomyocyte differentiation. CD144 (VE-cadherin)can be used as a marker of endothelial cells. Smooth muscle actin (SMA)can be used as a marker of smooth muscle cells. MLC2v can be used as amarker of ventricular muscle cells. MLC2a, which is expressed on bothimmature ventricular muscle cells and atrial muscle cells, can be usedas a marker for those cell types. Additionally, sarcolipin, which isspecifically expressed in atrial muscle cells, can be used as a markerfor atrial muscle cells. Phospholamban, which is expressed predominantlyin the ventricles and, to a lesser extent, in the atria, can also beused as a marker. Hairy-related transcription factor 1 (HRT1), alsocalled Hey1, which is expressed in atrial cardiomyocytes, can be used asa marker for atrial cardiomyocytes. HRT2 (Hey2), which is expressed inventricular cardiomyocytes, can be used as a marker for ventricularcardiomyocytes. In addition, IRX4 has a ventricular-restrictedexpression pattern during all stages of development, and thus can beused as a ventricular lineage marker. In summary, the genes expressed inthe ventricles, and thus which are appropriate ventricular markers, are:MLC2v, IRX4 and HRT2, while genes expressed in the atria, and thus whichare appropriate atrial markers are: MLC2a, HRT1, Sarcolipin and ANF(atrial natriuretic factor). The preferred marker of ventriculardifferentiation is MLC2v.

Clonal Populations of Human Cardiac Ventricular Progenitor Cells

In another aspect, the invention provides methods for obtaining a clonalpopulation of human cardiac ventricular progenitor cells, as well asisolated clonal populations of such progenitors. The invention allowsfor the expansion and propagation of the cardiac ventricular progenitorcells such that a clonal population of a billion or more cells can beachieved. The ability to clonally expand the NRP1+ cardiac ventricularprogenitor cells to such large numbers is a necessary feature forsuccessful use of these cells in vivo to enhance cardiac function, sincesuch a use requires on the order of a billion or more cells.

Accordingly, in another aspect, the invention provides a method forobtaining a clonal population of human cardiac ventricular progenitorcells, the method comprising:

isolating a single NRP1+ human cardiac ventricular progenitor cell; and

culturing the single NRP1+ human cardiac ventricular progenitor cellunder conditions such that the cell is expanded to at least 1×10⁹ cellsto thereby obtain a clonal population of human cardiac ventricularprogenitor cells.

In a preferred embodiment, the single NRP1+ human cardiac ventricularprogenitor cell is Islet 1 positive, Nkx2.5 negative and flk1 negativeat the time of initial culture. As described further in the Examples,such a single cell can be obtained at approximately day 6 of the cultureunder conditions that promote the generation of cardiomyogenicprogenitors. The clonal population of human cardiac ventricularprogenitors can be further cultured and differentiated in vitro suchthat the cells express the ventricular maker MLC2v.

Preferably, the single NRP1+ human cardiac ventricular progenitor cellis isolated by fluorescence activated cell sorting. Alternatively, thecell can be isolated by MACS or by other cell sorting methods known inthe art and/or described herein.

Preferably, the single NRP1+ human cardiac ventricular progenitor cellis isolated using one or more agents reactive with NRP1, such as ananti-NRP1 antibody or other agent reactive with NRP1 as describedhereinbefore.

In other embodiments, the clonal population of human cardiac ventricularprogenitor cells is NRP1+ and positive for at least one second marker ofHVPs (e.g., Isl1, FZD4, LIFR, FGFR3, TNFSF9). Such double-positive cellscan be isolated and clonally expanded as described herein before usingboth an agent reactive with NRP1 and an agent reactive with the secondmarker.

In a preferred embodiment, the single NRP1+ human cardiac ventricularprogenitor cell is cultured in Cardiac Progenitor Culture (CPC) medium,as described hereinbefore.

In a preferred embodiment, the single NRP1+ human cardiac ventricularprogenitor cell is cultured under conditions such that the cell isbiased toward ventricular differentiation. Preferred culture conditionsinclude culture in CPC medium.

In various embodiments, the single NRP1+ human cardiac ventricularprogenitor cell can be expanded to at least 1×10⁹ cells, at least 2×10⁹cells, at least 3×10⁹ cells, at least 4×10⁹ cells, at least 5×10⁹ cells,at least 6×10⁹ cells, at least 7×10⁹ cells, at least 8×10⁹ cells, atleast 9×10⁹ cells or at least 10×10⁹ cells.

Accordingly, the invention also provides a clonal population of at least1×10⁹ NRP1+ human cardiac ventricular progenitor cells, which areobtainable or obtained by the methods of the invention for obtaining aclonal population of human cardiac ventricular progenitor cells. Invarious embodiments, the clonal population of NRP1+ human cardiacventricular progenitor cells comprises at least 1×10⁹ cells, at least2×10⁹ cells, at least 3×10⁹ cells, at least 4×10⁹ cells, at least 5×10⁹cells, at least 6×10⁹ cells, at least 7×10⁹ cells, at least 8×10⁹ cells,at least 9×10⁹ cells or at least 10×10⁹ cells. Differentiation of theprogenitor cells to the ventricular lineage in vitro can be achieved byculture under conditions described herein for biasing toward theventricular lineage. Furthermore, transplantation of the cardiacventricular progenitor cells in vivo leads to ventriculardifferentiation in vivo.

The invention also provides pharmaceutical compositions comprising theclonal population of cardiac ventricular progenitor cells. Thepharmaceutical compositions typically are sterile and can comprisebuffers, media, excipients and the like suitable for pharmaceuticaladministration. In one embodiment, the pharmaceutical compositioncomprising the clonal population is formulated onto a three dimensional(3D) matrix. Compositions formulated onto a 3D matrix are particularlypreferred for formation of a heart muscle cell patch that can betransplanted in vivo for heart muscle repair. Furthermore, thecompositions can be formulated into two dimensional (2D) sheets ofcells, such as a muscular thin film (MTF) as described in Domian, I. J.et al. (2009) Science 326:426-429. Such 2D sheets of cell tissue alsocan be used in the formation of a heart muscle cell patch that can betransplanted in vivo for heart muscle repair.

Generation of Human Ventricular Progenitors (HVPs)

Prior to isolation by the aforementioned methods, and optionallyobtaining a clonal population by the aforementioned methods, anon-clonal population of human ventricular progenitors (HVPs) can beobtained by culture of human pluripotent stem cells (hPSCs) underappropriate culture conditions to generate the HVPs. An exemplary set ofculture conditions, and suitable starting cells, is described in detailin Example 1 and Example 10, also referred to herein as the HumanVentricular Progenitor Generation (HVPG) protocol. Suitable hPSCstarting cells include induced pluripotent stem cells (iPSC) and humanembryonic stem cells, such as ES cell lines. For the protocol,Wnt/β-catenin signaling first is activated in the hPSCs, followed by anincubation period, followed by inhibition of Wnt/β-catenin signaling.Wnt/β-catenin signaling activation is achieved by incubation with a Gsk3inhibitor, preferably CHIR98014 (CAS 556813-39-9; commercially availablefrom, e.g., Selleckchem). Wnt/β-catenin signaling inhibition is achievedby incubation with a Porcn inhibitor, preferably Wnt-C59 (CAS1243243-89-1; commercially available from, e.g., Selleckchem or Tocris).The Gsk3 inhibitor is used to promote cardiac mesodermaldifferentiation, whereas the Porcn inhibitor is used to enhanceventricular progenitor differentiation from mesoderm cells.

Accordingly, in another aspect, the invention provides a method ofgenerating human ventricular progenitors (HVPs) comprising culturinghuman pluripotent stems cells (hPSCs) in a medium comprising a Gsk3inhibitor, preferably CHIR98014, for at least 24 hours, more preferablyfor 2 days or 3 days, followed by culturing the hPSCs in a mediumcomprising a Porcn inhibitor, preferably Wnt-C59 (and lacking the Gsk3inhibitor), for at least 48 hours such that HVPs are generated.Experiments showed that after 24-hour treatment with CHIR-98014, morethan 99% of hPSCs expressed the mesoderm marker Brachyury, and threedays later after treatment with CHIR-98014, more than 95% ofdifferentiatated cells expressed Mesp1, which marks the cardiacmesoderm. Furthermore, 48-hour treatment with Wnt-C59 enhancedventricular progenitor differentiation from mesoderm cells.

Accordingly, with regard to timing of the use of the Gsk3 and Porcninhibitors, typically, at day 0 of culture, the hPSCs are cultured withthe Gsk3 inhibitor, at day 3 of culture the medium is changed to removethe Gsk3 inhibitor and the cells are then cultured with media containingthe Porcn inhibitor through day 5 of culture. HVP generation is optimalbetween days 5 and 7 (inclusive) in culture and peaks at day 6 ofculture. Other non-limiting, exemplary details on culture conditions andtiming of the use of the Gsk3 and Porcn inhibitors are described indetail in Examples 1 and 10.

In Vivo Tissue Engineering

In vivo transplantation studies described in Example 6 and 7 in whichthe human ventricular progenitors (HVPs) were transplanted under thekidney capsule in nude mice document the ability of the HVPs tospontaneously assemble into a large wall of mature, functional, humanventricular muscle on the surface of the kidney capsule. Vascularizationoccurs via a paracrine pathway by calling the murine vasculature to theventricular muscle wall, while a matrix is generated via a cellautonomous pathway from the progenitors themselves. In vivointra-myocardial transplantation studies described in Example 8 in whichthe HVPs were transplanted into the normal murine heart document thatthe HVPs spontaneously migrate to the epicardial surface, where theyexpand, subsequently differentiate, and mature into a wall of humanventricular muscle on the surface of the epicardium. Taken together,these studies show that human ventriculogenesis can occur via acompletely cell autonomous pathway in vivo via purified HVPs, therebyallowing their use in organ-on-organ in vivo tissue engineering.

The human ventricular myocardium has a limited capacity forregeneration, most of which is lost after 10 years of age (Bergmann, O.et al. (2015) Cell 161:1566-1575). As such, new strategies to generateheart muscle repair, regeneration, and tissue engineering approachesduring cardiac injury have been a subject of intense investigation inregenerative biology and medicine (Sahara, M. et al. (2015) EMBO J.34:710-738; Segers, V. F. M. and Lee, R. T. (2008) Nature 451:937-942).Given the need to achieve coordinated vascularization and matrixformation during tissue engineering of any solid organ, the assumptionhas been that the formation of an intact 3-D solid organ in vivo willultimately require the addition of vascular cells and/or conduits, aswell as biomaterials and/or decellularized matrix that will allowalignment and the generation of contractile force (Forbes, S. J. andRosenthal, N. (2014) Nature Med. 20:857-869; Harrison, R. H. et al.(2014) Tissue Eng. Part B Rev. 20:1-16). The complexity of adding thesevarious components to achieve the formation of a functional solid organhas confounded attempts to reduce this to clinical practice (Webber, M.J. et al. (2014) Ann. Biomed. Eng. 43:641-656). Although hPSCs holdgreat promise, to date, it has not been possible to build a pure,vascularized, fully functional, and mature 3-D human ventricular muscleorgan in vivo on the surface of a heart in any mammalian system(Vunjak-Novakovic, G. et al. (2011) Annu. Rev. Biomed. Eng. 13:245-267).

The ability of generate billions of purified HVPs from a renewablesource of either human ES or iPS cell lines represent a new approach tothe generation of functional ventricular muscle in the setting ofadvanced heart failure. The progenitors can be delivered byintramyocardial injection and then self-migrate to the epicardialsurface where they expand and differentiate, losing progenitor markers.Over the course of several week, the cells exit the cell cycle, andproceed to form adult rod-shaped cells that display several independentmarkers of mature ventricular myocardium including the formation of Ttubules, catecholamine responsiveness, loss of automaticity, adult rodshaped conformation with aligned sarcomenric structures, and the abilityto generate force that is comparable to other heart muscle patchesderived from hPSCs differentiated cardiomyocytes (Tulloch, N. L. et al.(2011) Circ. Res. 109:47-59). The scalability of this cell autonomouspathway has allowed the ectopic generation of human ventricular musclethat has a combined thickness in excess of 1.5 cm in thickness,approaching levels that correspond to the human ventricular free wall(Basavarajaiah, S. et al. (2007) Br. J. Sports Med. 41:784-788).

The ability to migrate to the epicardial niche, the site of most of theadult heart progenitors at later stages, is a unique feature of HVPs,and mimics the normal niche of these cells during expansion of theventricular compact zone during ventriculogenesis. Previous studies haveshown that the generation of acute ischemic injury and a breakdown invascular permeability are a pre-requisite for the grafting of relativelysmall numbers of ES cell derived cardiomyocytes into injured myocardium(van Laake, L. W. et al. (2007) Stem Cell Res. 1:9-24; Laflamme, M. A.et al. (2007) Nat. Biotechnol. 25:1015-1024), and even then the survivalrate is low (<5%) (Laflamme, M. A. and Murry, C. E. (2011) Nature473:326-335; Laflamme, M. A. et al. (2005) Am. J. Pathol. 167:663-671).The ability of intra-myocardial HVPs to form an extensive ventricularpatch on the epicardial surface in the absence of acute ischemic injuryprovides a new therapeutic strategy for dilated cardiomyopathy withoutthe need for additional biomaterials, cells, or transfer of exogenousgenes and/or RNAs.

The ability to form a 3-D ventricular muscle wall on the epicardialsurface of the in vivo normal heart is a unique feature of theventricular progenitors as later stage progenitors do not display theability for the formation of three-dimensional ventricular tissue ineither the cardiac or non-cardiac context, emphasizing the importance ofgenerating a committed ventricular lineage as well as purifying thespecific ventricular progenitor at a specific stage ofventriculogenesis.

Accordingly, the invention provides methods for generating humanventricular tissue in vivo using the HVPs described herein. In oneembodiment, the method comprises transplanting the NRP1+ progenitorsinto an organ of a non-human animal and allowing the progenitors to growin vivo such that human ventricular tissue is generated. Preferably, thenon-human animal is immunodeficient such that it cannot mount an immuneresponse against the human progenitor cells. In one embodiment, thenon-human animal is a mouse, such as an immunodeficient NOD.Cg-PrkdcscidIl2rgtm 1Wjl/SzJ mouse or an immunodeficient SCID-beige mouse(commercially available from Charles River France). In one embodiment,the organ is a kidney (e.g., the cells are transplanted under the kidneycapsule). In another embodiment, the organ is a heart. In variousembodiments, at least 1×10⁶ cells, at least 2×10⁶ cells, at least 3×10⁶cells, at least 4×10⁶ cells, at least 5×10⁶ cells, at least 1×10⁷ cells,at least 5×10⁷ cells, at least 1×10⁸ cells, at least 1×10⁹ cells aretransplanted.

To obtain HVPs for transplantation, human pluripotent stem cells (hPSCs)can be cultured in vitro under conditions leading to the generation ofHVPs, as described herein (referred to herein as the HVPG protocol).Regarding the timing of transplanting HVPs post in-vitro culture, foroptimal ventricular tissue generation the cells should be transplantedat a stage that can be defined based on the cellular markers expressedby the HVPs at the time of transplantation, determined at days post thestart of culture, which is defined as day 0 of the HVPG protocol. In oneembodiment, the cells are transplanted after the peak of cardiacmesoderm formation, which can be defined as peak expression of themesodermal marker MESP1. Typically, MESP1 expression is between day 2and day 4 of culture (inclusive) and peaks at day 3. In one embodiment,the cells are transplanted at the time corresponding to peak Islet-1expression. Typically, Islet 1 is expressed between day 4 to day 8 ofculture (inclusive) and peaks at day 6 of culture. In one embodiment,the cells are transplanted before the peak of NKX2.5 expression.Typically, NKX2.5 expression starts at day 6 of culture, peaks at day 10of culture and is then maintained afterwards. In one embodiment, thecells are transplanted prior to the peak expression of the downstreamgenes MEF-2 and TBX-1. Typically, these downstream genes are expressedbetween day 5 and day 15 of culture (inclusive) and peaks at day 8 ofculture. In one embodiment, the cells are transplanted prior to theexpression of differentiated contractile protein genes. Typically, theexpression of contractile protein genes (including TNNT2 and MYH6)starts from day 10 of culture onward. In certain embodiments, the cellsare transplanted at a time when two, three or four of the aforementionedmarker patterns are present. In another embodiment, the cells aretransplanted at a time when all five of the aforementioned markerpatterns are present. In one embodiment, the cells are transplantedbetween day 4 to day 8 (inclusive) of culture. In a more preferredembodiment, the cells are transplanted between day 5 to day 7(inclusive) of culture. In the most preferred embodiment, the cells aretransplanted on day 6 of culture.

The transplanted cells can be allowed to grow in the non-human animalfor a suitable period time to allow for the generation of the desiredsize, amount or thickness of ventricular tissue. In various embodiments,the cells are allowed to grow for one week, two weeks, one month, twomonths, three months, four months, five months or six months. The methodcan further comprise harvesting ventricular tissue from the non-humananimal after growth of the cells and differentiation into ventriculartissue.

Methods of Enhancing Cardiac Function

The cardiac ventricular progenitor cells of the invention can be used invivo to enhance cardiac function by transplanting the cells directlyinto the heart. It has now been shown that the NRP1+ progenitors havethe capacity to differentiate into all three types of cardiac lineagecells (cardiac myocytes, endothelial cells and smooth muscle cells) (seeExample 3). Furthermore, when cultured under conditions that bias towardthe ventricular lineage, the NRP1+ progenitors have now been shown toadopt a predominantly ventricular muscle phenotype when transplantedinto the natural ventricle environment in vivo, demonstrating that theseprogenitor cells “recognize” the ventricular environment and respond anddifferentiate appropriately in vivo. Since damage to the ventricularenvironment is largely responsible for the impaired cardiac function incardiac diseases and disorders, the ability to restore ventricularmuscle cells using the ventricular progenitor cells of the inventionrepresents a significant advance in the art.

Accordingly, in another aspect, the invention provides a method ofenhancing cardiac function in a subject, the method comprisingadministering a pharmaceutical composition comprising the clonalpopulation of NRP1+ cardiac ventricular progenitor cells of theinvention to the subject. Preferably, the clonal population isadministered directly into the heart of the subject. More preferably,the clonal population is administered directly into a ventricular regionof the heart of the subject. In one embodiment, the pharmaceuticalcomposition administered to the subject comprises the clonal populationformulated onto a three dimensional matrix.

The methods of the invention for enhancing cardiac function in a subjectcan be used in a variety of clinical situations involving damage to theheart or reduced or impaired cardiac function. Non-limiting examples ofsuch clinical situations include a subject who has suffered a myocardialinfarction and a subject who has a congenital heart disorder. Thus, inanother aspect, the invention provides a method of treating acardiovascular condition, disease or disorder in a subject, the methodcomprising administering a pharmaceutical composition comprising theclonal population of NRP1+ cardiac ventricular progenitor cells of theinvention to the subject. A therapeutically effective amount of cardiacventricular progenitor cells can be administered for the treatment of acardiovascular condition, disease or disorder. Examples of preferredcardiovascular conditions, diseases or disorders include coronary arterydisease and acute coronary syndrome.

Methods of Use of Cardiac Ventricular Progenitor Cells In Vitro

The cardiac ventricular progenitor cells of the invention can be used invitro in the study of various aspects of cardiac maturation anddifferentiation, in particular in identifying the cells signalingpathways and biological mediators involved in the process of cardiacmaturation and differentiation.

Furthermore, since the NRP1+ cardiac ventricular progenitor cells of theinvention are committed to the cardiac lineage and, moreover, are biasedtoward ventricular differentiation, these progenitor cells also areuseful for evaluating the cardiac toxicity of test compounds. Allpotential new drugs and therapeutics must be evaluated for theirtoxicity to cardiac cells, before they can be deemed safe for use inhumans. Thus, the ability to assess cardiac toxicity in an in vitroculture system is very advantageous. Accordingly, in another aspect, theinvention provides a method of screening for cardiac toxicity of testcompound, the method comprising:

providing NRP1+ human cardiac ventricular progenitor cells;

contacting the cells with the test compound; and

measuring toxicity of the test compound for the cells,

wherein toxicity of the test compound for the cells indicates cardiactoxicity of the test compound.

In a preferred embodiment, the NRP1+ human cardiac ventricularprogenitor cells are provided by isolating the cells according to themethods described herein. In a particularly preferred embodiment, thecells are isolated by separating NRP1+ cells from a cell culturecomprising cardiac progenitor cells using an anti-NRP1 antibody.Preferably, the cells are isolated using FACS or MACS as describedherein. In yet another embodiment, the NRP1+ human cardiac ventricularprogenitor cells are further cultured and differentiation into MLC2v+ventricular cells prior to contacting with the test compound.

The toxicity of the test compound for the cells can be measured by oneor more of a variety of different methods for assessing cell viabilityor other physiological functions. Preferably, the effect of the testcompound on cell viability is measured using a standard cell viabilityassay, wherein reduced cell viability in the presence of the testcompound is indicative of cardiac toxicity of the test compound.Additionally or alternatively, cell growth can be measured. Additionallyor alternatively, other indicators of physiological functions can bemeasured, such as cell adhesion, cell signaling, surface markerexpression, gene expression and the like. Similarly, a negative effectof the test compound on any of these indicators of physiologicalfunction is indicative of cardiac toxicity of the test compound.

The invention further provides a method of identifying a compound thatmodulates human cardiac ventricular progenitor cell differentiation, themethod comprising:

providing NRP1+ human cardiac ventricular progenitor cells;

culturing the cells in the presence or absence of a test compound;

measuring differentiation of the cells in the presence or absence of thetest compound; and

selecting a test compound that modulates human cardiac ventricularprogenitor cell differentiation, as compared to differentiation in theabsence of the test compound, to thereby identify a compound thatmodulates human cardiac ventricular progenitor cell differentiation.

In one embodiment, the test compound stimulates human cardiacventricular progenitor cell differentiation. In another embodiment, thetest compound inhibits human cardiac ventricular progenitor celldifferentiation. Differentiation of the cells can be measured by, forexample, measurement of the expression of differentiation markersappearing on the cultured cells over time, as described herein. In apreferred embodiment, the NRP1+ human cardiac ventricular progenitorcells are provided by isolating the cells according to the methodsdescribed herein. In a particularly preferred embodiment, the cells areisolated by separating NRP1+ cells from a cell culture comprisingcardiac progenitor cells using an anti-NRP1 antibody. Preferably, thecells are isolated using FACS or MACS as described herein.

The invention further provides a method of identifying a compound thatmodulates human ventricular cardiomyocyte function, the methodcomprising:

providing NRP1+ human cardiac ventricular progenitor cells;

culturing the cells in the presence or absence of a test compound underconditions that generate human ventricular cardiomyocytes;

measuring function of the human ventricular cardiomyocytes in thepresence or absence of the test compound; and

selecting a test compound that modulates human ventricular cardiomyocytefunction, as compared to function in the absence of the test compound,to thereby identify a compound that modulates human ventricularcardiomyoctye function.

In one embodiment, the test compound stimulates human ventricularcardiomyocyte function. In another embodiment, the test compoundinhibits human ventricular cardiomyocyte function. Function of the cellscan be measured by measurement of any suitable indicator of ventricularcell function, including but not limited to, for example, formation of Ttubules, acquisition of adult-rod shaped ventricular cardiomyocytes, andability to generate force in response to electrical stimulation.Suitable assays for measuring such indicators of ventricular cellfunction are known in the art. In a preferred embodiment, the NRP1+human cardiac ventricular progenitor cells are provided by isolating thecells according to the methods described herein. In a particularlypreferred embodiment, the cells are isolated by separating NRP1+ cellsfrom a cell culture comprising cardiac progenitor cells using ananti-NRP1 antibody. Preferably, the cells are isolated using FACS orMACS as described herein.

In Vivo Animal Models Using Human Ventricular Progenitor Cells

The development of human iPS and ES cell based models of cardiac diseasehas opened new horizons in cardiovascular drug development anddiscovery. However, to date, these systems have had the limitations ofbeing based on 2D structures in cultured cell systems. In addition, thefetal and immature properties of the cells limit their utility andfidelity to the adult heart. Human cardiac disease, in particular heartfailure, is a complex, multifactorial, multi-organ disease, that isinfluenced by environmental, hormonal, and other key organs that areknown sites for therapeutic targets, such as the kidney. The ability tobuild a mature functional human ventricular organ either ectopically oron the surface of the intact normal murine heart opens up a new in vivomodel system to allow studies that normally could only be assayed on amature human ventricular muscle chamber, such as ventriculararrhythmias, generation of contractile force, fibrosis, and thepotential for regeneration. Accordingly, the option to study humancardiac disease outside of the in vitro tissue culture systems, anddirectly in the context of heart failure in vivo, is now clearlypossible.

Thus, the human ventricular progenitor cells also can be used to createanimal models that allow for in vivo assessment of human cardiac tissuefunction and for in vivo screening of compounds, such as to determinethe cardiac toxicity of a test compound in vivo or to identify compoundsthat modulate human cardiac tissue differentiation or function in vivo.Accordingly, the invention provides methods for testing the effects oftest compounds on human ventricular tissue in vivo using the HVPsdescribed herein. In one embodiment, the method comprises:

transplanting NRP1+ human ventricular progenitors into an organ of anon-human animal;

allowing the progenitors to grow in vivo such that human ventriculartissue is generated;

administering a test compound to the non-human animal; and

evaluating the effect of the test compound on the human ventriculartissue in the non-human animal.

In another embodiment, the method comprises:

administering a test compound to a non-human animal, wherein thenon-human animal comprises NRP1+ human ventricular progenitorstransplanted into an organ of the non-human animal; and

evaluating the effect of the test compound on the NRP1+ humanventricular progenitors in the non-human animal.

In one embodiment, the cardiac toxicity of the test compound isevaluated, for example by measuring the effect of the test compound onthe viability of the human ventricular tissue or the NRP1+ humanventricular progenitors in the non-human animal (as compared to theviability of the tissue or progenitors in the absence of the testcompound). Cell viability can be assessed by standard methods known inthe art.

In another embodiment, the ability of a test compound to modulatecardiac differentiation can be evaluated, for example by measuring theeffect of the test compound on the differentiation of the humanventricular tissue or NRP1+ progenitors in the non-human animal (ascompared to the differentiation of the tissue or progenitors in theabsence of the test compound). Differentiation of the cells can bemeasured by, for example, measurement of the expression ofdifferentiation markers appearing on the cells over time.

In another embodiment, the ability of a test compound to modulatecardiac function can be evaluated, for example by measuring the effectof the test compound on the function of the human ventricular tissue orNRP1+ human progenitors in the non-human animal (as compared to thefunction of the tissue or progenitors in the absence of the testcompound). Function of the tissue or progenitors can be measured bymeasurement of any suitable indicator of ventricular cell function,including but not limited to, for example, formation of T tubules,acquisition of adult-rod shaped ventricular cardiomyocytes, and abilityto generate force in response to electrical stimulation. Suitable assaysfor measuring such indicators of ventricular cell function are known inthe art.

Preferably, the non-human animal is immunodeficient such that it cannotmount an immune response against the human progenitor cells. In oneembodiment, the non-human animal is a mouse, such as an immunodeficientNOD.Cg-Prkdcscid Il2rgtm 1Wjl/SzJ mouse or an immunodeficient SCID-beigemouse (commercially available from Charles River France). In oneembodiment, the organ is a kidney (e.g., the cells are transplantedunder the kidney capsule). In another embodiment, the organ is a heart.In various embodiments, at least 1×10⁶ cells, at least 2×10⁶ cells, atleast 3×10⁶ cells, at least 4×10⁶ cells, at least 5×10⁶ cells, at least1×10⁷ cells, at least 5×10⁷ cells, at least 1×10⁸ cells, at least 1×10⁹cells are transplanted.

To create the animal models, HVPs for transplantation can be obtained asdescribed above by culturing of hPSCs in vitro under conditions leadingto the generation of HVPs. Regarding the timing of transplanting HVPspost in-vitro culture, for optimal ventricular tissue generation thecells should be transplanted at a stage that can be defined based on thecellular markers expressed by the HVPs at the time of transplantation,determined at days post the start of culture, which is defined as day 0of the HVPG protocol. In one embodiment, the cells are transplantedafter the peak of cardiac mesoderm formation, which can be defined aspeak expression of the mesodermal marker MESP1. Typically, MESP1expression is between day 2 and day 4 of culture (inclusive) and peaksat day 3. In one embodiment, the cells are transplanted at the timecorresponding to peak Islet-1 expression. Typically, Islet 1 isexpressed between day 4 to day 8 of culture (inclusive) and peaks at day6 of culture. In one embodiment, the cells are transplanted before thepeak of NKX2.5 expression. Typically, NKX2.5 expression starts at day 6of culture, peaks at day 10 of culture and is then maintainedafterwards. In one embodiment, the cells are transplanted prior to thepeak expression of the downstream genes MEF-2 and TBX-1. Typically,these downstream genes are expressed between day 5 and day 15 of culture(inclusive) and peaks at day 8 of culture. In one embodiment, the cellsare transplanted prior to the expression of differentiated contractileprotein genes. Typically, the expression of contractile protein genes(including TNNT2 and MYH6) starts from day 10 of culture onward. Incertain embodiments, the cells are transplanted at a time when two,three or four of the aforementioned marker patterns are present. Inanother embodiment, the cells are transplanted at a time when all fiveof the aforementioned marker patterns are present. In one embodiment,the cells are transplanted between day 4 to day 8 (inclusive) ofculture. In a more preferred embodiment, the cells are transplantedbetween day 5 to day 7 (inclusive) of culture. In the most preferredembodiment, the cells are transplanted on day 6 of culture.

The transplanted cells can be allowed to grow in the non-human animalfor a suitable period time to allow for the generation of the desiredsize, amount or thickness of ventricular tissue, prior to administrationof the test compound(s). In various embodiments, the cells are allowedto grow for one week, two weeks. one month, two months, three months,four months, five months or six months.

The present invention is further illustrated by the following examples,which should not be construed as further limiting. The contents offigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1 Generation of Human Isl1+ Cardiomyogenic ProgenitorCells by Modulation of Wnt Signaling in Human Pluripotent Stem Cells

Temporal modulation of canonical Wnt signaling has been shown to besufficient to generate functional cardiomyocytes at high yield andpurity from numerous hPSC lines (Lian, X. et al. (2012) Proc. Natl.Acad. Sci. USA109:E1848-1857; Lian, X. et al. (2013) Nat. Protoc.8:162-175). In this approach, Wnt/β-catenin signaling first is activatedin the hPSCs, followed by an incubation period, followed by inhibitionof Wnt/β-catenin signaling. In the originally published protocol,Wnt/β-catenin signaling activation was achieved by incubation with theGsk3 inhibitor CHIR99021 (GSK-3α, IC₅₀=10 nM; GSK-3, β IC₅₀=6.7 nM) andWnt/β-catenin signaling inhibition was achieved by incubation with thePorcn inhibitor IWP2 (IC₅₀=27 nM). Because we used Gsk3 inhibitor andWnt production inhibitor for cardiac differentiation, this protocol wastermed GiWi protocol. To improve the efficiency of the original protocoland reduce the potential side effects of the small molecules used in theoriginal protocol, a second generation protocol was developed that usesanother set of small molecules with higher inhibition potency. In thissecond generation GiWi protocol, Wnt/β-catenin signaling activation wasachieved by incubation with the Gsk3 inhibitor CHIR98014 (CAS556813-39-9; commercially available from, e.g., Selleckchem) (GSK-3α,IC₅₀=0.65 nM; GSK-3, β IC50=0.58 nM) and Wnt/β-catenin signalinginhibition was achieved by incubation with the Porcn inhibitor Wnt-C59(CAS 1243243-89-1; commercially available from, e.g., Selleckchem orTocris) (IC₅₀=74 pM). The Gsk3 inhibitor CHIR98014 was used to promotecardiac mesodermal differentiation, whereas the Porcn inhibitor Wnt-C59was used to enhance ventricular progenitor differentiation from mesodermcells.

For cardiomyocyte differentiation via the use of these small molecules,hPSCs were maintained on Matrigel (BD Biosciences) coated plates(Corning) in E8 medium (described in Chen, G. et al. (2011) NatureMethods, 8:424-429; commercially available; STEMCELL Technologies) ormTeSR1 medium (commercially available; STEMCELL Technologies). SuitablehPSCs include induced pluripotent stem cells (iPSCs) such as 19-11-1,19-9-7 or 6-9-9 cells (Yu, J. et al. (2009) Science, 324:797-801) andhuman embryonic stem cells (hESCs), such as ES03 (WiCell ResearchInstitute) and H9 cells (Thomson, J. A. et al. (1998) Science,282:1145-1147).

hPSCs maintained on a Matrigel-coated surface in mTeSR1 medium weredissociated into single cells with Accutase (Life Technologies) at 37°C. for 5 minutes and then seeded onto a Matrigel-coated cell culturedish at 100,000-200,000 cells/cm² in mTeSR1 medium supplemented with 5μM ROCK inhibitor Y-27632 (Selleckchem)(day −2) for 24 hours. Cells werethen cultured in mTeSR1, changed daily. At day 0, cells were thentreated with 1 μM Gsk3 inhibitor CHIR98014 (Selleckchem) for 24 hours(day 0 to day 1) in RPMI/B27-ins (500 ml RPMI with 10 ml B27 supplementwithout insulin). The medium was then changed to the correspondingmedium containing 2 μM the Porcn inhibitor Wnt-C59 (Selleckchem) at day3, which was then removed during the medium change on day 5. Cells weremaintained in RPMI/B27 (stock solution: 500 ml RMPI medium+10 ml B27supplement) starting from day 7, with the medium changed every threedays. This exemplary culturing protocol for generating cardiomyogenicprogenitor cells is illustrated schematically in FIG. 1.

Flow cytometry and immunostaining were preformed to examine theexpression of particular lineage markers. After 24 hour treatment withCHIR-98014, more than 99% of the hPSCs expressed the mesoderm markerBrachyury. Three days after treatment with CHIR-98014, more than 95% ofdifferentiated cells expressed Mesp1, which marks the cardiac mesoderm.The culture protocol not only allowed the cells to synchronouslydifferentiate into the cardiac mesodermal lineage, but also reproduciblygenerated more than 90% of ventricular myocytes after 14 days ofdifferentitation, as determined by cTnT flow cytometry andelectrophysiology analysis.

To further assess cardiac differentiation of the hPSCs over time,Western blot analysis was performed on days 0-7 and dll to examine theexpression of Isl1 and Nkx2.5 (cardiomyogenic progenitor markers) andcTnl (a cardiac myocyte marker). Cells were lysed in

M-PER Mammalian Protein Extraction Reagent (Pierce) in the presence ofHalt Protease and Phosphatase Inhibitor Cocktail (Pierce). Proteins wereseparated by 10% Tris-Glycine SDS/PAGE (Invitrogen) under denaturingconditions and transferred to a nitrocellulose membrane. After blockingwith 5% dried milk in TBST, the membrane was incubated with primaryantibody overnight at 4° C. The membrane was then washed, incubated withan anti-mouse/rabbit peroxidase-conjugated secondary antibody at roomtemperature for 1 hour, and developed by SuperSignal chemiluminescence(Pierce). The results are shown in FIG. 2. During cardiacdifferentiation of hPSCs, Isl1 expression started on day 4 and increasedto its maximum expression on day 6, whereas NKx2.5 only started toexpress on day 6 and reached its maximum expression after day 10.Cardiomyoctes (cTnI+ cells) were not induced until day 11 ofdifferentiation.

In addition, immunostaining of the day 6 cells was performed for Isl1expression. Cells were fixed with 4% formaldehyde for 15 minutes at roomtemperature and then stained with primary (anti-Isl1) and secondaryantibodies in PBS plus 0.4% Triton X-100 and 5% non-fat dry milk(Bio-Rad). Nuclei were stained with Gold Anti-fade Reagent with DAPI(Invitrogen). An epifluorescence microscope (Leica DM IRB) with aQlmaging® Retiga 4000R camera was used for imaging analysis. The resultsshowed substantial numbers of Isl1+ cells.

Flow cytometry analysis of day 6 cells for Isl1 expression also wasperformed. Cells were dissociated into single cells with Accutase for 10minutes and then fixed with 1% paraformaldehyde for 20 minutes at roomtemperature and stained with primary and secondary antibodies in PBS0.1% Triton X-100 and 0.5% BSA. Data were collected on a FACSCaliberflow cytometer (Beckton Dickinson) and analyzed using FloJo. Theresults, shown in FIG. 3, showed that more than 95% of cells expressedIsl1 at this stage.

In summary, this example provides a protocol for human ventricularprogenitor generation (HVPG protocol) that allows for the large-scaleproduction of billions of Isl1+ human HPVs efficiently within 6 days.

Example 2 Identification of Jagged 1 as a Cell Surface Marker of CardiacProgenitor Cells

To profile the transcriptional changes that occur during the cardiacdifferentiation process at a genome-scale level, RNA sequencing(RNA-seq) was performed at different time points followingdifferentiation to build cardiac development transcriptional landscapes.We performed RNA-seq experiments on day 0 to day 7 samples, as well asday 19 and day 35 samples (two independent biological replicates pertime point). Two batches of RNA-seq (100 bp and 50 bp read length) wereperformed using the illumine Hiseq 2000 platform. In total, 20 sampleswere examined. Bowtie and Tophat were used to map our reads into areference human genome (hg19) and we calculate each gene expression(annotation of the genes according to Refseq) using RPKM method (Readsper kilobase transcript per million reads). Differentiation of hPSCs tocardiomyocytes involves five major cell types: pluripotent stem cells(day 0), mesoderm progenitors (day 1 to day 2), cardiac mesoderm cells(day 3 to day 4), heart field progenitors (day 5, day 6 and day 7), andcardiomyocytes (day 10 after).

Molecular mRNA analysis of cardiac differentiation from hPSCs using theHVPG protocol revealed dynamic changes in gene expression, withdown-regulation of the pluripotency markers OCT4, NANOG and SOX2 duringdifferentiation. Induction of the primitive streak-like genes T andMIXL1 occurred within the first 24 hours following CHIR-98014 addition,and was followed by upregulation of the cardiac mesodermal marker MESP1on day 2 and day 3. Expression of the cardiac muscle markers TNNT2,TNNC1, MYL2, MYL7, MYH6, MYH7 and IRX4 was detected at later stage ofdifferentiation (after day 10).

By this analysis, genes enriched at each differentiation stage,including mesoderm cells, cardiac progenitors and cardiomyocytes, wereidentified. Mesoderm cells, which are related to day 1 differentiatedcells, express brachyury. We identified potential surface markers formesoderm cells, including: FZD10, CD48, CD1D, CD8B, IL15RA, TNFRSF1B,TNFSF13, ICOSLG, SEMA7A, SLC3A2, SDC1, HLA-A. Through similar analysis,we also identified surface markers for cardiac mesoderm mespl positivecells, including: CXCR4, ANPEP, ITGA5, TNFRSF9, FZD2, CD1D, CD177,ACVRL1, ICAM1, L1CAM, NGFR, ABCG2, FZD7, TNFRSF13C, TNFRSF1B.

Consistent with western blot analysis, ISL1 mRNA was expressed as earlyas day 4 and peaked on day 5, one day before its protein expressionreached its peak. On day 5 of differentiation (the cardiac progenitorstage, isll mRNA expression maximum on day 5, isl1 protein expressionmaximum on day 6), the day 5 enriched genes were compared with ananti-CD antibody array (a panel of 350 known CD antibodies) and a numberof potential cell-surface protein markers were identified. We identifiedmany cell-surface proteins expressed at this stage, including: FZD4,JAG1, PDGFRA, LIFR (CD118), TNFSF9, FGFR3.

The cell surface protein Jagged 1 (JAG1) and Frizzled 4 (FZD4) wereselected for further analysis. Jagged 1 expression was further studiedas described below and in Examples 3 and 4. Frizzled 4 expression wasfurther studied as described in Example 5.

Firstly, the expression of Isl1 and Jag1 was profiled using the doublestaining flow cytometry technique. Flow cytometric analysis was carriedout essentially as described in Example 1, using anti-Isl1 and anti-Jag1antibodies for double staining. The results are shown in FIG. 4. Jagged1 expression was found to trace the expression of Islet 1 and on day 6of differentiation, all of the Islet 1 positive cells also expressedJagged 1, and vice versa. Because of the co-expression pattern of thesetwo markers, a Jagged 1 antibody was used to enrich the 94.1% Islet 1+cells differentiated population to 99.8% purity of Islet1+Jagged1+cells.

It also was confirmed that Islet 1 is an earlier developmental gene thanthe Nkx2.5 gene using double immunostaining of ISL1 and NKX2.5expression in HVPs. The purified HVPs uniformly express the ISL1 gene,but at this stage, only a few of the cells started to express Nkx2.5.

Furthermore, immunostaining with both anti-Isl1 and anti-Jag 1 wasperformed, essentially as described in Example 1, on week 4 human fetalheart tissue, neonatal heart tissue and 8-year old heart tissue. Theresults revealed that in the in vivo fetal heart, all of the Islet 1positive cells also expressed Jagged 1. However, the neonatal heart and8-year old heart did not express Islet 1 or Jagged 1. In the ventricleof week 4 human fetal heart, cardiac Troponin T (cTnT) staining revealedvisible sarcomere structures. In addition, over 50% of ventricular cellsin the week 4 fetal heart expressed both Islet1 and Jagged1, which wasmarkedly decreased during subsequent maturation, with the loss ofexpression of both Islet1 and Jagged1 in the ventricular muscle cells ofthe human neonatal hearts.

The above-described experiments demonstrate that Jagged 1 is a cellsurface marker for Islet 1 positive cardiomyogenic progenitor cells.

Example 3 Clonal Differentiation of Isl1+Jag1+ Cardiac Progenitor Cells

To characterize the clonal differentiation potential of Isl1+Jag1+cells, cardiomyogenic progenitor cells were generated by the culturingprotocol described in Example 1, and one single Isl1+Jag1+ cell wasseeded into one well of a Matrigel-coated 48-well plate. Cells werepurified with antibody of Jag1 and then one single cell was seeded intoone well. The single cells were then cultured for 3 weeks in CardiacProgenitor Culture (CPC) medium (advanced DMEM/F12 supplemented with 2.5mM GlutaMAX, 100 μg/ml Vitamin C, 20% Knockout Serum Replacement).

Immunostaining of the 3-week differentiation cell population was thenperformed with three antibodies: cardiac troponin I (cTn1) forcardiomyocytes, CD144 (VE-cadherin) for endothelial cells and smoothmuscle actin (SMA) for smooth muscle cells. The results showed that thesingle cell-cultured, Isl1+Jag1+ cells gave rise to cTnI positive andSMA positive cells, but not VE-cadherin positive endothelial cells,indicating these generated Islet1+ cells are heart muscle progenitorsthat have limited differentiation potential to endothelial lineages.Purified Islet1+Jagged1+ cells differentiated with the HVPG protocolfrom human induced pluripotent stem cells (iPSC 19-9-11 line) alsoshowed similar in vitro differentiation potential and predominantlydifferentiate to cTnI+SMA+ cells, but not VE-cadherin+ cells. Over thecourse of several weeks, the cells expressed the ventricular specificmarker MLC2v, indicating that the initial ISL1+ subset was alreadycommitted to the ventricular cell fate. Because of the limited vasculardifferentiation potential of Islet1+ cells generated using the HVPGprotocol, these generated Islet1+ cells might represent a distinctprogenitor population from the previously reported KDR+ population(Yang, L. et al. (2008) Nature 453:524-528) or multipotent ISL1+ cells(Bu, L. et al. (2009) Nature 460:113-117; Moretti, A. et al. (2006) Cell127:1151-1165), which can give rise to all three lineages ofcardiovascular cells.

These results demonstrated that the Isl1+Jag1+ cardiomyogenic progenitorcells can be successfully cultured in vitro from a single cell to asignificantly expanded cell population (1×10⁹ cells or greater) thatcontains all three types of cardiac lineage cells, with a predominanceof cardiomyocytes. Furthermore, these cells can be cultured in vitro forextended periods of time, for at least 2-3 weeks, and even for months(e.g., six months or more). Since the cardiomyogenic progenitor cellsgradually differentiate into cardiomyocytes, which do not proliferate, aculture period of approximately 2-3 weeks is preferred.

Example 4 In Vivo Developmental Potential of Isl1+Jag1+ CardiacProgenitor Cells

The ES03 human embryonic stem cell (hESC) line (obtained from WiCellResearch Institute) expresses green fluorescent protein (GFP) driven bythe cardiac-specific cTnT promoter. ES03 cells were used to generateIsl1+Jag1+ cardiomyogenic progenitor cells using the culturing protocoldescribed in Example 1. The Isl1+Jag1+ cardiomyogenic progenitor cellswere transplanted into the hearts of severe combined immunodeficient(SCID) beige mice to document their developmental potential in vivo.

Briefly, Isl1+Jag1+ cells were injected (1,000,000 cells per recipient)directly into the left ventricular wall of NOD/SCID-gamma mice in anopen-chest procedure. Hearts were harvested 2-3 weeks post-surgery,fixed in 1% PFA and sectioned at 10 μm (n=12). Histological analyses ofthe hearts of the transplanted mice revealed the presence of GFP+ donorcells, detected by epifluorescence and by staining with an anti-GFPantibody, demonstrating that the Isl1+Jag1+ cardiomyogenic progenitorcells were capable of differentiating into cardiomyocytes whentransplanted in vivo.

The Isl1+Jag1+ cardiomyogenic progenitor cells were also transplanteddirectly into infarcted hearts of SCID beige mice (“injured mice”), ascompared to similarly transplanted normal mice. When analyzed two weekslater, injured mice transplanted with the Isl1+Jag1+ cardiomyogenicprogenitor cells had a larger graft size than the normal mice similarlytransplanted, demonstrating the cardiomyocyte regeneration capacity ofthe Isl1+Jag1+ cardiomyogenic progenitor cells in vivo.

Example 5 Identification of Frizzled 4 as a Cell Surface Marker ofCardiac Progenitor Cells

As described in Example 2, Frizzled 4 (FZD4) was identified by RNA-seqanalysis as being expressed in cardiac progenitor cells. Thus, toconfirm FZD4 as a cell surface marker of cardiac progenitor cells, FZD4expression was assessed during cardiac differentiation via Western blotanalysis. The results, as shown in FIG. 5, demonstrated that FZD4 wasnot express in pluripotent stem cells and the first 3 daysdifferentiated cells. However, FZD4 started to express on day 4 andmaximize its expression on day 5 of expression.

In order to quantify the co-expression pattern of FZD4 and Isl1 at thesingle cell level, FACS analysis was performed. As shown in FIG. 6, onday 5 of differentiation, more than 83% of cells express both isll andFZD4, demonstrating that FZD4 is a cell surface marker for isll positivecells during cardiac progenitor differentiation using the GiWi protocol.

In order to confirm that both JAG1 and FZD4 were indeed co-expressedwith ISL1 on the human ventricular progenitor cells, tripleimmunofluorescence analysis of day 6 differentiated cells from hPSCs wasperformed with antibodies to Islet 1, Jagged 1 and Frizzled 4. Thetriple staining experiment demonstrated that Isl1+ cells expressed bothJagged 1 and Frizzled 4.

Example 6 Human Ventricular Progenitors (HPVs) Generate a 3-DVentricular Heart Muscle Organ In Vivo

The building of the ventricular heart muscle chamber is one of the mostcritical and earliest steps during human organogenesis, and requires aseries of coordinated steps, including migration, proliferation,vascularization, assembly, and matrix alignment. To test the capacity ofHVPs to drive ventriculogenesis in vivo, we transplanted purified HVPsor unpurified HVPs (92.0±1.9% ISL1+) under the kidney capsule ofimmunocompromised mice. After 2 months post-transplantation, animalstransplanted with unpurified HVPs formed tumors, resulting in a tumorformation efficiency of 100% (100%, 4/4), whereas animals transplantedwith purified HVPs did not form any tumors (0%, 0/10).

The engrafted kidneys with purified HVPs were further assayed forhistological analysis. Hematoxylin and Eosin (H&E) staining revealed anorgan that exceeded 0.5 cm in length with more than 1 mm thickness onthe surface of the mouse kidney, and that uniformly expressed theventricular specific marker MLC2v (O'Brien, T. X. et al. (1993) Proc.Natl. Acad. Sci. USA 90:5157-5161). The resulting human muscle organ wasfully vascularized and red blood cells could be detected in the bloodvessels. Analysis of cTnT, MLC2v, and MLC2a immunostaining furtherrevealed that the transplanted HVPs not only differentiated into cardiacmuscle cells (cTnT+ cells), but also further mature to become MLC2v+ventricular myocytes that are negative for MLC2a expression. Theresulting ventricular muscle organ is fully vascularized by murinederived vascular cells, consistent with the notion that itsvascularization occurred via paracrine cues derived from the HVPs.

The blood vessel structured was revealed by immunostaining analysis ofantibodies directed against VE-cadherin and smooth muscle actinexpression. In addition, using a human specific monoclonal lamininantibody targeting laminin γ-1 chain, the HVPs secreted their own humanlaminin as their extracellular matrix (the mouse kidney region isnegative for human laminin immunostaining). In addition, we found humanfibronectin expression is restricted to areas near the blood vesselsusing a monoclonal human fibronectin antibody.

To assess the capacity of late stage cardiac cells to driveventriculogenesis, NKX2.5+ cells (day 10 after differentiation) weretransplanted under the kidney capsule of immunocompromised NSG mice. Atthree weeks post-transplantation, animals transplanted with NKX2.5+cells did not form any visible human muscle graft, indicating that HVPslose their ability for in vivo ventriculogenesis following peak Islet-1expression.

Taken together, these studies indicate that the HVPs can synthesize andrelease their own cardiac laminin-derived matrix, as well as fibronectinwhich serves to stabilize the vasculature to the nascent ventricularorgan.

Example 7 HVPs Create a Mature, Functioning Ventricular Muscle Organ InVivo Via a Cell Autonomous Pathway

One of the critical limitations for the utility of hPSCs for studies ofhuman cardiac biology and disease is their lack of maturity andpersistence of expression of fetal isoforms. To determine if the HVPderived organs could become functional mature ventricular muscle, longterm transplantation studies were performed followed by detailedanalyses of a panel of well accepted features of adult ventricularmyocardium including formation of T tubules (Brette, F. and Orchard, C.(2003) Circ. Res. 92:1182-1192; Marks, A. R. (2013) J. Clin. Invest.123:46-52), ability to generate force comparable to other studies ofengineered ventricular tissue, loss of automaticity, and acquisition ofadult-rod shaped ventricular cardiomyocytes.

After 5 months post-transplantation of purified HVPs, no tumors formedin all of our animals. Animals were sacrificed and the engrafted kidneyswere removed for further analysis. The 5-month human graft was ahemisphere structure with the radius of 0.4 cm (diameter of 0.8 cm). Thevolume for the 5-month human graft was around 0.13 cm³ for one kidney, avolume that suggests feasibility for generating human ventricular musclethat achieves a thickness comparable to the in vivo human adult heart.Rod-shaped mature human ventricular myocytes were observed in the humanmuscle organ. In addition, muscle trips taken from our mature humanmuscle organ generated forces (0.36±0.04 mN) in response to electricstimulation and increased their force generation after treatment with aβ-adrenergic agonist isoprenaline (0.51±0.02 mN, p<0.05 compared tocontrol). Taken together, these studies indicate that the HVPs arecapable of generating a fully functional, mature human ventricularmuscle organ in vivo via a cell autonomous pathway, i.e., without theaddition of other cells, genes, matrix proteins, or biomaterials.

Example 8 HVPs Migrate Towards an Epicardial Niche and SpontaneouslyForm a Human Ventricular Muscle Patch on the Surface of a Normal MurineHeart In Vivo

The epicardium is a known niche for heart progenitors, driving thegrowth of the ventricular chamber during compact zone expansion, as wellas serving as a home to adult epicardial progenitors that can expandafter myocardial injury and that can drive vasculogenesis in response toknown vascular cell fate switches, such as VEGF (Giordano, F. J. et al.(2001) Proc. Natl. Acad. Sci. USA 98:5780-5785; Masters, M. and Riley,P. R. (2014) Stem Cell Res. 13:683-692; Zangi, L. et al. (2013) Nat.Biotechnol. 31:898-907). To determine if the HVPs might migratespontaneously to the epicardial surface of the normal heart, purifiedgreen fluorescent protein (GFP)-labeled HVPs were injectedintra-myocardially into the hearts of immunocompromised mice. After oneweek or one month post-transplantation, animals were sacrificed and theengrafted hearts were removed for histology. After one weekpost-transplantation, the majority of GFP+ cells were retained in themyocardium. However, almost all the GFP+ cells migrated to theepicardium after one month post-transplantation. In addition, GFP+ cellswere ISL1+ and Ki67+ after one week post-transplantation.

In order to trace the differentiation potential of Islet1+ cells, thepurified ISL1+JAG1+ cells generated from a cTnT promoter driven greenfluorescent protein (GFP)-expressing hESC line (H9-cTnT-GFP) weretransplanted into the hearts of severe combined immunodeficient (SCID)beige mice to document their developmental potential in vivo. One monthafter transplantation of Isl1+Jag1+ cells directly into the ventricle ofthe hearts of SCID beige mice, Hematoxylin and eosin staining revealed ahuman muscle strip graft present in the epicardium of the murine heart.In addition, immunohistological analyses revealed the presence of GFP+donor cells detected by epifluorescence and by staining with an anti-GFPantibody. More importantly, when analysed with antibodies of MLC2v andMLC2a, the grafted human muscle strip is positive for MLC2v (100% ofcells+), and negative for the atrial marker MLC2a, indicating thetransplanted ISL1+ cells not only further differentiated to cardiacmuscle cells, but also became ventricular muscle cells.

Taken together, these studies indicate that the HVPs can migrate to anepicarial niche, where they expand, and subsequently differentiate in toa homogenous ventricular muscle patch, again without the addition ofexogenous cells, genes, matrices, or biomaterials.

Example 9 Additional Experimental Materials and Methods

In this example, additional details on the experimental materials andmethods used in Examples 1-8 are provided.

Maintenance of hPSCs

hESCs (ES03, H9) and human iPSCs (19-9-11) were maintained on Matrigel(BD Biosciences) coated plates in mTeSR1 medium (STEMCELL Technologies)according to previous published methods (Lian, X. et al. (2013) Nat.Proc. 8:162-175; Lian, X. et al. (2013) Stem Cells 31:447-457).

Human Ventricular Progenitor Generation (HVPG) Protocol

hPSCs maintained on a Matrigel-coated surface in mTeSR1 were dissociatedinto single cells with Accutase at 37° C. for 10 min and then seededonto a Matrigel-coated cell culture dish at 100,000-200,000 cell/cm² inmTeSR1 supplemented with 5 μM ROCK inhibitor Y-27632 (day −2) for 24hours. At day −1, cells were cultured in mTeSR1. At day 0, cells weretreated with 1 μM CHIR-98014 (Selleckchem) in RPMI supplemented with B27minus insulin (RPMI/B27-ins) for 24 hours (day 0 to day 1), which wasthen removed during the medium change on day 1. At day 3, half of themedium was changed to the RPMI/B27-ins medium containing 2 μM Wnt-C59(Selleckchem), which was then removed during the medium change on day 5.At day 6, cells were dissociated into single cells and purified withanti-JAG1 or anti-FZD4 antibody.

RNA-seq Library Construction

RNA was isolated (RNeasy Mini kit, Qiagen), quantified (Qubit RNA AssayKit, Life Technologies) and quality controlled (BioAnalyzer 2100,Agilent). RNA (800 ng) from each sample was used as input for theIllumina TruSeq mRNA Sample Prep Kit v2 (Illumina) and sequencinglibraries were created according to the manufacturer's protocol.Briefly, poly-A containing mRNA molecules were purified using poly-Toligo-attached magnetic beads. Following purification, the mRNA wasfragmented and copied into first strand complementary DNA using randomprimers and reverse transcriptase. Second strand cDNA synthesis was thendone using DNA polymerase I and RNase H. The cDNA was ligated toadapters and enriched with PCR to create the final cDNA library. Thelibrary was pooled and sequenced on a HiSeq 2000 (Illumina) instrumentper the manufacturer's instructions.

RNA-seq Data Processing

The RNA-seq reads were trimmed and mapped to the hg19 reference usingTophat 2. On average, approximately 23 million reads were generated persample, and 76% of these reads were uniquely mapped. Expression levelsfor each gene were quantified using the python script rpkmforgenes andannotated using RefSeq. Genes without at least one sample with at leastten reads were removed from the analysis. Principle Component Analysisand heatmaps were constructed using the R and Gene-E respectively.

Transplantation

Aliquots of 2 million purified HVPs were collected into an eppendorftube. Cells were spun down, and the supernatant was discarded. Each tubeof cells was transplanted under the kidney capsule, orintra-myocardially injected into the heart of the immunodeficient mice,NOD.Cg-Prkdcscid Il2rgtm 1Wjl/SzJ or SCID-Beige respectively (CharlesRiver France), following a previously described protocol (Shultz, L. D.et al. (2005) J. Immunol. 174:6477-6489). Engrafted Kidneys or heartsare harvested at various time intervals for histological andphysiological analysis.

Flow Cytometry

Cells were dissociated into single cells with Accutase for 10 min andthen fixed with 1% paraformaldehyde for 20 min at room temperature andstained with primary and secondary antibodies in PBS plus 0.1% TritonX-100 and 0.5% BSA. Data were collected on a FACSCaliber flow cytometer(Beckton Dickinson) and analyzed using FlowJo.

Immunostaining

Cells were fixed with 4% paraformaldehyde for 15 min at room temperatureand then stained with primary and secondary antibodies in PBS plus 0.4%Triton X-100 and 5% non-fat dry milk (Bio-Rad). Nuclei were stained withGold Anti-fade Reagent with DAPI (Invitrogen). An epifluorescencemicroscope and a confocal microscope (ZEISS, LSM 700) were used forimaging analysis.

Western Blot Analysis

Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce)in the presence of Halt Protease and Phosphatase Inhibitor Cocktail(Pierce). Proteins were separated by 10% Tris-Glycine SDS/PAGE(Invitrogen) under denaturing conditions and transferred to anitrocellulose membrane. After blocking with 5% dried milk in TBST, themembrane was incubated with primary antibody overnight at 4° C. Themembrane was then washed, incubated with an anti-mouse/rabbitperoxidase-conjugated secondary antibody at room temperature for 1 hour,and developed by SuperSignal chemiluminescence (Pierce).

Electrophysiology (Patch Clamping)

Beating ventricular myocyte clusters were microdissected and replatedonto glass coverslips before recording. Action potential activity wasassessed using borosilicate glass pipettes (4-5 M Ohm resistance) filledwith intracellular solution consisting of 120 mM K D-gluconate, 25 mMKCl, 4 mM MgATP, 2 mM NaGTP, 4 mM Na2-phospho-creatin, 10 mM EGTA, 1 mMCaCl2, and 10 mM HEPES (pH 7.4 adjusted with HCl at 25° C.). Culturedcardiomyocytes seeded on coverslip dishes were submerged inextracellular solution (Tyrode's solution) containing 140 mM NaCl, 5.4mM KCl, 1 mM MgCl2, 10 mM glucose, 1.8 mM CaCl2, and 10 mM HEPES (pH 7.4adjusted with NaOH at 25° C.). Spontaneous action potentials wererecorded at 37° C. using patch clamp technique (whole-cell, currentclamp configuration) performed using a Multiclamp 700B amplifier(Molecular Devices, CA, USA) software low-pass filtered at 1 kHz,digitized and stored using a Digidata 1322A and Clampex 9.6 software(Molecular Devices, CA, USA).

Statistics

Data are presented as mean±standard error of the mean (SEM). Statisticalsignificance was determined by Student's t-test (two-tail) between twogroups. P<0.05 was considered statistically significant.

Example 10 Xeno-Free Human Ventricular Progenitor DifferentiationProtocol

In this example, an alternative differentiation protocol fordifferentiation of human ventricular progenitors is provided, whichutilizes a defined, xeno-free culture medium, Essential 8. The Essential8 medium was developed for growth and expansion of human pluripotentstem cells (hPSCs) and is described further in Chen, G. et al. (2011)Nat. Methods 8:424-429 (referred to therein as “E8” medium).

hPSCs maintained on a Vitronectin (or Laminin 521)-coated surface inEssential 8 medium were dissociated into single cells with Versenesolution at 37° C. for 10 min and then seeded onto a Vitronectin (orLaminin 521)-coated cell culture dish at 100,000-200,000 cell/cm² inEssential 8 medium supplemented with 5 μM ROCK inhibitor Y-27632 (day−2) for 24 hours. At day −1, cells were cultured in Essential 8 medium.At day 0, cells were treated with 0.5 μM CHIR-98014 in RPMI for 24 hours(day 0 to day 1), which was then removed during the medium change onday 1. At day 3, half of the medium was changed to the RPMI mediumcontaining 0.5 μM Wnt-C59, which was then removed during the mediumchange on day 5. At day 6, cells (human ventricular progenitors) weredissociated into single cells and purified with anti-JAG1 or anti-FZD4antibody. Alternatively cells are purified with anti-LIFR or anti-FGFR3antibody.

Example 11 Angiogenic Markers for Engraftable Human VentricularProgenitor Cells

In this example, genes in the angiogenic family that are expressed inhuman ventricular progenitor cells (HVPs) were identified. HVPs weregenerated as described in Examples 1 or 10 and RNA sequencing (RNA-seq)was performed at different time points following differentiation asdescribed in Example 1. Cluster analysis of gene expression profiles atdifferent time points during HVP differentiation identifiedstage-specific signature genes. These genes were clusteredhierarchically on the basis of the similarity of their expressionprofiles. First, genes showing expression in four different categorieswere identified: (i) cell surface expression; (ii) co-expression withIslet 1; (iii) high expression on day 5 of differentiation; and (iv)high d5/d0 ratio. This analysis confirmed the cell surface markers forHVPs of: JAG1, FZD4, FGFR3, LIFR (CD118) and TNFSF9. Next, from thissame population of HVPs that identified the cell surface markers, geneontogeny searches were performed to identify angiogenic family genesthat were expressed in this population of HVPs, to thereby identify agene fingerprint profile that identifies genes critical for cellengraftment.

Statistically, Pearson's correlation with Isl1 expression was used toidentify those angiogenic genes whose expression in the HVPs bestcorrelated with Isl1 expression. Table 1 below lists the angiogenicgenes that correlate with Isl1 expression with a Pearson's correlationof 0.50 or higher.

TABLE 1 Angiogenic genes expressed in HVPs with a Pearson Correlationwith Isl1 Expression of 0.50 or greater Pearson's Correlation with GeneAngiogenic genes (GO:0001525) Isl1 Expression FGF10 fibroblast growthfactor 10 0.98 PRKD1 protein kinase D1 0.95 CCBE1 collagen and calciumbinding EGF domains 1 0.94 PDGFRA platelet-derived growth factorreceptor, alpha polypeptide 0.94 EPHB2 EPH receptor B2 0.92 GATA2 GATAbinding protein 2 0.92 NTRK1 neurotrophic tyrosine kinase, receptor,type 1 0.92 PTGIS prostaglandin I2 (prostacyclin) synthase 0.87 BMPERBMP binding endothelial regulator 0.85 BMP4 bone morphogenetic protein 40.84 C1GALT1 core 1 synthase, glycoprotein-N-acetylgalactosamine 3- 0.84beta-galactosyltransferase 1 MEIS1 Meis homeobox 1 0.83 TBX1 T-box 10.83 PKNOX1 PBX/knotted 1 homeobox 1 0.83 ID1 inhibitor of DNA binding1, dominant negative helix-loop- 0.82 helix protein TCF21 transcriptionfactor 21 0.82 HEY1 hes-related family bHLH transcription factor withYRPW 0.80 motif 1 HOXB3 homeobox B3 0.78 JAG1 jagged 1 0.75 HGFhepatocyte growth factor (hepapoietin A; scatter factor) 0.74 IL6interleukin 6 0.74 GHRL ghrelin/obestatin prepropeptide 0.73 IHH indianhedgehog 0.70 SRPK2 SRSF protein kinase 2 0.70 GATA6 GATA bindingprotein 6 0.69 HAND1 heart and neural crest derivatives expressed 1 0.69AMOT angiomotin 0.69 NRP2 neuropilin 2 0.65 PTEN phosphatase and tensinhomolog 0.65 SEMA3E sema domain, immunoglobulin domain (Ig), short basic0.64 domain, secreted, (semaphorin) 3E APOLD1 apolipoprotein L domaincontaining 1 0.62 SETD2 SET domain containing 2 0.62 DAB2IP DAB2interacting protein 0.61 KDR kinase insert domain receptor 0.60 PGFplacental growth factor 0.60 EMP2 epithelial membrane protein 2 0.59TAL1 T-cell acute lymphocytic leukemia 1 0.58 ACVR1 activin A receptor,type I 0.58 HIPK2 homeodomain interacting protein kinase 2 0.56 CSPG4chondroitin sulfate proteoglycan 4 0.55 TNFAIP3 tumor necrosis factor,alpha-induced protein 3 0.55 NRP1 neuropilin 1 0.55 NFATC4 nuclearfactor of activated T-cells, cytoplasmic, 0.54 calcineurin-dependent 4CDC42 cell division cycle 42 0.54 ANGPTL4 angiopoietin-like 4 0.53 BCAS3breast carcinoma amplified sequence 3 0.53 HIPK1 homeodomain interactingprotein kinase 1 0.53 NRXN3 neurexin 3 0.52 FZD5 frizzled class receptor5 0.52 HHEX hematopoietically expressed homeobox 0.50Table 2 below lists the angiogenic genes that correlate with Isl1expression with a Pearson's correlation of 0.49-0.00.

TABLE 2 Angiogenic genes expressed in HVPs with a Pearson Correlationwith Isl1 Expression of 0.49 to 0.00 Pearson's Correlation with GeneAngiogenic genes (GO:0001525) Isl1 Expression ACVRL1 activin A receptortype Il-like 1 0.49 ENPEP glutamyl aminopeptidase (aminopeptidase A)0.49 EFNA1 ephrin-A1 0.49 CHRNA7 cholinergic receptor, nicotinic, alpha7 (neuronal) 0.49 TMEM100 transmembrane protein 100 0.48 NOS3 nitricoxide synthase 3 (endothelial cell) 0.47 LEF1 lymphoid enhancer-bindingfactor 1 0.47 NRXN1 neurexin 1 0.46 EPHB3 EPH receptor B3 0.44 ROCK1Rho-associated, coiled-coil containing protein kinase 1 0.42 NF1neurofibromin 1 0.42 CYSLTR2 cysteinyl leukotriene receptor 2 0.42 FGFR2fibroblast growth factor receptor 2 0.41 GATA4 GATA binding protein 40.40 FMNL3 formin-like 3 0.40 C3 complement component 3 0.40 WASF2 WASprotein family, member 2 0.40 CALCRL calcitonin receptor-like 0.39 HIF1Ahypoxia inducible factor 1, alpha subunit (basic helix-loop- 0.39 helixtranscription factor) VEGFA vascular endothelial growth factor A 0.39KRIT1 KRIT1, ankyrin repeat containing 0.39 CDH13 cadherin 13 0.39COL18A1 collagen, type XVIII, alpha 1 0.39 STK4 serine/threonine kinase4 0.38 C5 complement component 5 0.38 HDAC7 histone deacetylase 7 0.38ANGPT2 angiopoietin 2 0.38 PLCG1 phospholipase C, gamma 1 0.37 EDNRAendothelin receptor type A 0.35 TGFB2 transforming growth factor, beta 20.35 HAND2 heart and neural crest derivatives expressed 2 0.35 CD34 CD34molecule 0.35 BTG1 B-cell translocation gene 1, anti-proliferative 0.34TGFBR1 transforming growth factor, beta receptor 1 0.33 FGFR1 fibroblastgrowth factor receptor 1 0.33 FN1 fibronectin 1 0.31 TWIST1 twist familybHLH transcription factor 1 0.31 ELK3 ELK3, ETS-domain protein (SRFaccessory protein 2) 0.30 THSD7A thrombospondin, type I, domaincontaining 7A 0.30 RGCC regulator of cell cycle 0.30 PLCD1 phospholipaseC, delta 1 0.29 SPARC secreted protein, acidic, cysteine-rich(osteonectin) 0.29 TBX20 T-box 20 0.28 PIK3CAphosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic 0.27 subunitalpha MMRN2 multimerin 2 0.27 FOXO4 forkhead box O4 0.26 RAMP2 receptor(G protein-coupled) activity modifying protein 2 0.25 FLT1 fms-relatedtyrosine kinase 1 0.25 ADRB2 adrenoceptor beta 2, surface 0.25 SLC12A6solute carrier family 12 (potassium/chloride transporter), 0.25 member 6ADM adrenomedullin 0.25 NPPB natriuretic peptide B 0.24 SPINK5 serinepeptidase inhibitor, Kazal type 5 0.24 MAPK14 mitogen-activated proteinkinase 14 0.24 MMP2 matrix metallopeptidase 2 0.24 PTPRM proteintyrosine phosphatase, receptor type, M 0.23 OVOL2 ovo-like zinc finger 20.23 CTNNB1 catenin (cadherin-associated protein), beta 1, 88 kDa 0.22OTULIN OTU deubiquitinase with linear linkage specificity 0.21 B4GALT1UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, 0.21 polypeptide 1PDGFRB platelet-derived growth factor receptor, beta polypeptide 0.20 F3coagulation factor III (thromboplastin, tissue factor) 0.20 PRKCAprotein kinase C, alpha 0.20 LRP5 low density lipoproteinreceptor-related protein 5 0.20 MAP3K7 mitogen-activated protein kinasekinase kinase 7 0.20 NRCAM neuronal cell adhesion molecule 0.19 MAP2K5mitogen-activated protein kinase kinase 5 0.18 S1PR1sphingosine-1-phosphate receptor 1 0.18 NFATC3 nuclear factor ofactivated T-cells, cytoplasmic, 0.18 calcineurin-dependent 3 TSPAN12tetraspanin 12 0.18 LAMA5 laminin, alpha 5 0.17 LOXL2 lysyl oxidase-like2 0.17 ANGPT1 angiopoietin 1 0.17 GTF2I general transcription factor IIi0.16 E2F8 E2F transcription factor 8 0.16 PDE3B phosphodiesterase 3B,cGMP-inhibited 0.15 SHB Src homology 2 domain containing adaptor proteinB 0.14 MYH9 myosin, heavy chain 9, non-muscle 0.14 FZD8 frizzled classreceptor 8 0.14 NOV nephroblastoma overexpressed 0.14 SH2D2A SH2 domaincontaining 2A 0.14 FGF8 fibroblast growth factor 8 (androgen-induced)0.13 TIE1 tyrosine kinase with immunoglobulin-like and EGF-like 0.13domains 1 EGLN1 egl-9 family hypoxia-inducible factor 1 0.12 RORARAR-related orphan receptor A 0.11 MFGE8 milk fat globule-EGF factor 8protein 0.11 ARHGAP24 Rho GTPase activating protein 24 0.10 ITGA5integrin, alpha 5 (fibronectin receptor, alpha polypeptide) 0.10 PARVAparvin, alpha 0.10 ADIPOR2 adiponectin receptor 2 0.09 NPR1 natriureticpeptide receptor 1 0.09 ITGB1 integrin, beta 1 (fibronectin receptor,beta polypeptide, 0.09 antigen CD29 includes MDF2, MSK12) HIF3A hypoxiainducible factor 3, alpha subunit 0.08 EPAS1 endothelial PAS domainprotein 1 0.08 FOXC2 forkhead box C2 0.07 ANXA2 annexin A2 0.06 RBM15RNA binding motif protein 15 0.06 PITX2 paired-like homeodomain 2 0.06FOXC1 forkhead box C1 0.06 SRF serum response factor 0.06 ECSCRendothelial cell surface expressed chemotaxis and 0.05 apoptosisregulator SOX17 SRY (sex determining region Y)-box 17 0.04 HDAC5 histonedeacetylase 5 0.04 LRG1 leucine-rich alpha-2-glycoprotein 1 0.04 ADAM8ADAM metallopeptidase domain 8 0.03 UBP1 upstream binding protein 1(LBP-1a) 0.02 VASH1 vasohibin 1 0.02 ANXA3 annexin A3 0.01 RRAS relatedRAS viral (r-ras) oncogene homolog 0.01 TYMP thymidine phosphorylase0.01 PRCP prolylcarboxypeptidase (angiotensinase C) 0.01 SEMA5A semadomain, seven thrombospondin repeats (type 1 and 0.00 type 1 -like),transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5AGREM1 gremlin 1, DAN family BMP antagonist 0.00

Angiogenic genes whose expression negatively correlated with Isl1expression in the HVPs were also identified. Table 3 below lists theangiogenic genes that negatively correlate with Isl1 expression with aPearson's correlation of −0.50 or less.

TABLE 3 Angiogenic genes expressed in HVPs with a Pearson Correlationwith Isl1 Expression of −0.50 or less Pearson's Correlation with GeneAngiogenic genes (GO: 0001525) Isl1 Expression ETS1 v-ets avianerythroblastosis virus −0.50 E26 oncogene homolog 1 BAX BCL2-associatedX protein −0.50 XBP1 X-box binding protein 1 −0.52 TDGF1teratocarcinoma-derived −0.53 growth factor 1 C5AR1 complement component−0.53 5a receptor 1 EPHA1 EPH receptor A1 −0.53 HS6ST1 heparan sulfate6-O-sulfotransferase 1 −0.56 SHC1 SHC (Src homology 2 domain −0.56containing) transforming protein 1 SP100 SP100 nuclear antigen −0.58JAM3 junctional adhesion molecule 3 −0.58 CASP8 caspase 8,apoptosis-related −0.60 cysteine peptidase FLT4 fms-related tyrosinekinase 4 −0.60 SFRP2 secreted frizzled-related protein 2 −0.61 HPSEheparanase −0.61 BAK1 BCL2-antagonist/killer 1 −0.65 GPX1 glutathioneperoxidase 1 −0.65 VAV3 vav 3 guanine nucleotide exchange factor −0.70VAV2 vav 2 guanine nucleotide exchange factor −0.72 EGF epidermal growthfactor −0.72 ADAM15 ADAM metallopeptidase domain 15 −0.73 AGGF1angiogenic factor with G patch −0.76 and FHA domains 1Table 4 below lists the angiogenic genes that negatively correlate withIsl1 expression with a Pearson's correlation of −0.01 to −0.49.

TABLE 4 Angiogenic genes expressed in HVPs with a Pearson Correlationwith Isl1 Expression of −0.01 to −0.49 Pearson's Correlation with GeneAngiogenic genes (GO: 0001525) Isl1 Expression EIF2AK3 eukaryotictranslation initiation factor 2-alpha kinase 3 −0.01 ROCK2Rho-associated, coiled-coil containing protein kinase 2 −0.01 WNT5Awingless-type MMTV integration site family, member 5A −0.02 NR4A1nuclear receptor subfamily 4, group A, member 1 −0.02 CYP1B1 cytochromeP450, family 1, subfamily B, polypeptide 1 −0.02 PTK2 protein tyrosinekinase 2 −0.03 SFRP1 secreted frizzled-related protein 1 −0.04 STAT1signal transducer and activator of transcription 1, 91 kDa −0.04 ITGAVintegrin, alpha V −0.04 EPHB4 EPH receptor B4 −0.05 CYR61 cysteine-rich,angiogenic inducer, 61 −0.05 TEK TEK tyrosine kinase, endothelial −0.06COL15A1 collagen, type XV, alpha 1 −0.06 COL4A1 collagen, type IV, alpha1 −0.07 ANG angiogenin, ribonuclease, RNase A family, 5 −0.07 HSPB1 heatshock 27 kDa protein 1 −0.07 PLXND1 plexin D1 −0.08 HSPG2 heparansulfate proteoglycan 2 −0.09 VEGFC vascular endothelial growth factor C−0.09 SYNJ2BP synaptojanin 2 binding protein −0.09 THBS1 thrombospondin1 −0.09 CTGF connective tissue growth factor −0.10 ITGB3 integrin, beta3 (platelet glycoprotein IIIa, antigen CD61) −0.12 AAMPangio-associated, migratory cell protein −0.12 GJA5 gap junctionprotein, alpha 5, 40 kDa −0.12 PRKCB protein kinase C, beta −0.13 EGR3early growth response 3 −0.13 JMJD6 jumonji domain containing 6 −0.13TGFBI transforming growth factor, beta-induced, 68 kDa −0.14 SIRT1sirtuin 1 −0.14 ANGPTL3 angiopoietin-like 3 −0.14 ACKR3 atypicalchemokine receptor 3 −0.14 SAT1 spermidine/spermine N1-acetyltransferase1 −0.15 VEGFB vascular endothelial growth factor B −0.16 UTS2 urotensin2 −0.16 JUN jun proto-oncogene −0.16 TNFSF12 tumor necrosis factor(ligand) superfamily, member 12 −0.16 EGFL7 EGF-like-domain, multiple 7−0.17 MED1 mediator complex subunit 1 −0.17 SLIT2 slit guidance ligand 2−0.17 SERPINF1 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin,−0.18 pigment epithelium derived factor), member 1 NOTCH3 notch 3 −0.18FGF9 fibroblast growth factor 9 −0.19 DLL4 delta-like 4 (Drosophila)−0.19 CCL2 chemokine (C-C motif) ligand 2 −0.19 MMP14 matrixmetallopeptidase 14 (membrane-inserted) −0.19 TMPRSS6 transmembraneprotease, serine 6 −0.19 EPGN epithelial mitogen −0.20 RBPJrecombination signal binding protein for immunoglobulin −0.20 kappa Jregion COL4A2 collagen, type IV, alpha 2 −0.20 PRKD2 protein kinase D2−0.20 ALOX12 arachidonate 12-lipoxygenase −0.21 RNH1ribonuclease/angiogenin inhibitor 1 −0.21 APOH apolipoprotein H(beta-2-glycoprotein I) −0.21 CHI3L1 chitinase 3-like 1 (cartilageglycoprotein-39) −0.21 ESM1 endothelial cell-specific molecule 1 −0.22PTGS2 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H −0.22synthase and cyclooxygenase) ANPEP alanyl (membrane) aminopeptidase−0.22 LEMD3 LEM domain containing 3 −0.22 UTS2R urotensin 2 receptor−0.22 CIB1 calcium and integrin binding 1 (calmyrin) −0.22 ITGB1BP1integrin beta 1 binding protein 1 −0.22 AQP1 aquaporin 1 (Colton bloodgroup) −0.22 IL18 interleukin 18 −0.22 EPHA2 EPH receptor A2 −0.22 EPHB1EPH receptor B1 −0.22 AGT angiotensinogen (serpin peptidase inhibitor,clade A, −0.22 member 8) PLAU plasminogen activator, urokinase −0.22VEZF1 vascular endothelial zinc finger 1 −0.23 SPHK1 sphingosine kinase1 −0.23 SRPX2 sushi-repeat containing protein, X-linked 2 −0.23 PDCL3phosducin-like 3 −0.23 COL8A1 collagen, type VIII, alpha 1 −0.24 HDAC9histone deacetylase 9 −0.24 CTSH cathepsin H −0.24 EDN1 endothelin 1−0.24 CXCL8 chemokine (C—X—C motif) ligand 8 −0.24 ECM1 extracellularmatrix protein 1 −0.24 BRCA1 breast cancer 1, early onset −0.24 EFNB2ephrin-B2 −0.25 SERPINE1 serpin peptidase inhibitor, clade E (nexin,plasminogen −0.25 activator inhibitor type 1), member 1 SASH1 SAM andSH3 domain containing 1 −0.25 WNT7B wingless-type MMTV integration sitefamily, member 7B −0.25 RAMP1 receptor (G protein-coupled) activitymodifying protein 1 −0.26 SCG2 secretogranin II −0.26 COL8A2 collagen,type VIII, alpha 2 −0.26 SULF1 sulfatase 1 −0.26 CLIC4 chlorideintracellular channel 4 −0.26 FGF1 fibroblast growth factor 1 (acidic)−0.27 NODAL nodal growth differentiation factor −0.27 RASIP1 Rasinteracting protein 1 −0.28 RLN2 relaxin 2 −0.28 POFUT1 proteinO-fucosyltransferase 1 −0.28 FGF18 fibroblast growth factor 18 −0.28AIMP1 aminoacyl tRNA synthetase complex-interacting −0.28multifunctional protein 1 TGFBR2 transforming growth factor, betareceptor II (70/80 kDa) −0.28 RHOB ras homolog family member B −0.28GBX2 gastrulation brain homeobox 2 −0.28 ENPP2 ectonucleotidepyrophosphatase/phosphodiesterase 2 −0.29 MAPK7 mitogen-activatedprotein kinase 7 −0.30 PROK2 prokineticin 2 −0.30 E2F7 E2F transcriptionfactor 7 −0.30 ERAP1 endoplasmic reticulum aminopeptidase 1 −0.31 MTDHmetadherin −0.31 KLF5 Kruppel-like factor 5 (intestinal) −0.31 DICER1dicer 1, ribonuclease type III −0.32 LECT1 leukocyte cell derivedchemotaxin 1 −0.32 CX3CL1 chemokine (C—X3—C motif) ligand 1 −0.32 PTK2Bprotein tyrosine kinase 2 beta −0.33 SEMA4A sema domain, immunoglobulindomain (Ig), −0.34 transmembrane domain (TM) and short cytoplasmicdomain, (semaphorin) 4A ARHGAP22 Rho GTPase activating protein 22 −0.34RSPO3 R-spondin 3 −0.34 KLF4 Kruppel-like factor 4 (gut) −0.34 ROBO1roundabout guidance receptor 1 −0.34 GPLD1 glycosylphosphatidylinositolspecific phospholipase D1 −0.35 NUS1 NUS1 dehydrodolichyl diphosphatesynthase subunit −0.35 NRARP NOTCH-regulated ankyrin repeat protein−0.35 PDCD10 programmed cell death 10 −0.36 PF4 platelet factor 4 −0.36PRKX protein kinase, X-linked −0.36 PML promyelocytic leukemia −0.36ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, −0.36beta polypeptide TNFRSF12A tumor necrosis factor receptor superfamily,member 12A −0.36 ENG endoglin −0.37 THY1 Thy-1 cell surface antigen−0.37 FGF2 fibroblast growth factor 2 (basic) −0.37 CXCL12 chemokine(C—X—C motif) ligand 12 −0.37 CAV1 caveolin 1, caveolae protein, 22 kDa−0.38 PDGFA platelet-derived growth factor alpha polypeptide −0.38PNPLA6 patatin-like phospholipase domain containing 6 −0.38 PLCD3phospholipase C, delta 3 −0.38 DDAH1 dimethylargininedimethylaminohydrolase 1 −0.39 GNA13 guanine nucleotide binding protein(G protein), alpha 13 −0.39 ADM2 adrenomedullin 2 −0.39 HMOX1 hemeoxygenase 1 −0.40 MCAM melanoma cell adhesion molecule −0.41 RAPGEF3 Rapguanine nucleotide exchange factor (GEF) 3 −0.41 TNFAIP2 tumor necrosisfactor, alpha-induced protein 2 −0.41 HTATIP2 HIV-1 Tat interactiveprotein 2, 30 kDa −0.42 NCL nucleolin −0.42 ERBB2 erb-b2 receptortyrosine kinase 2 −0.43 NAA15 N(alpha)-acetyltransferase 15, NatAauxiliary subunit −0.43 ATPIF1 ATPase inhibitory factor 1 −0.43 THBS4thrombospondin 4 −0.43 SYK spleen tyrosine kinase −0.44 LIF leukemiainhibitory factor −0.44 THBS2 thrombospondin 2 −0.44 PPP1R16B proteinphosphatase 1, regulatory subunit 16B −0.44 NOTCH1 notch 1 −0.44 RUNX1runt-related transcription factor 1 −0.45 PDCD6 programmed cell death 6−0.45 VASH2 vasohibin 2 −0.45 GPI glucose-6-phosphate isomerase −0.46ZC3H12A zinc finger CCCH-type containing 12A −0.46 WARStryptophanyl-tRNA synthetase −0.46 HYAL1 hyaluronoglucosaminidase 1−0.47 PIK3CB phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic−0.47 subunit beta TNMD tenomodulin −0.49

Example 12 Extracellular Matrix Markers for Engraftable HumanVentricular Progenitor Cells

In this example, genes in the extracellular matrix family that areexpressed in human ventricular progenitor cells (HVPs) were identified.HVPs were generated as described in Examples 1 or 10 and RNA sequencing(RNA-seq) was performed at different time points followingdifferentiation as described in Example 1. Cluster analysis of geneexpression profiles at different time points during HVP differentiationidentified stage-specific signature genes. These genes were clusteredhierarchically on the basis of the similarity of their expressionprofiles. First, genes showing expression in four different categorieswere identified: (i) cell surface expression; (ii) co-expression withIslet 1; (iii) high expression on day 5 of differentiation; and (iv)high d5/d0 ratio. This analysis confirmed the cell surface markers forHVPs of: JAG1, FZD4, FGFR3, LIFR (CD118) and TNFSF9. Next, from thissame population of HVPs that identified the cell surface markers, geneontogeny searches were performed to identify extracellular matrix familygenes that were expressed in this population of HVPs, to therebyidentify a gene fingerprint profile that identifies genes critical forcell engraftment.

Statistically, Pearson's correlation with Isl1 expression was used toidentify those extracellular matrix genes whose expression in the HVPsbest correlated with Isl1 expression. Table 5 below lists theextracellular matrix genes that correlate with Isl1 expression with aPearson's correlation of 0.50 or higher.

TABLE 5 Extracellular matrix genes expressed in HVPs with a PearsonCorrelation with Isl1 Expression of 0.50 or greater Pearson'sCorrelation with Gene Extracellular matrix genes (GO: 0031012) Isl1Expression FGF10 fibroblast growth factor 10 0.98 SMOC1 SPARC relatedmodular calcium binding 1 0.97 CCBE1 collagen and calcium binding 0.94EGF domains 1 COL6A6 collagen, type VI, alpha 6 0.89 ADAMTS12 ADAMmetallopeptidase with 0.85 thrombospondin type 1 motif, 12 COL19A1collagen, type XIX, alpha 1 0.85 LAMA1 laminin, alpha 1 0.85 BMP4 bonemorphogenetic protein 4 0.84 FBLN7 fibulin 7 0.81 FBLN2 fibulin 2 0.81NDNF neuron-derived neurotrophic factor 0.80 HTRA1 HtrA serine peptidase1 0.80 HAPLN1 hyaluronan and proteoglycan 0.79 link protein 1 EMILIN1elastin microfibril interfacer 1 0.79 SPOCK3 sparc/osteonectin, cwcv andkazal- 0.76 like domains proteoglycan (testican) 3 PODNL1 podocan-like 10.73 IHH indian hedgehog 0.70 ACAN aggrecan 0.69 NID2 nidogen 2(osteonidogen) 0.69 COL4A6 collagen, type IV, alpha 6 0.68 LAMC1laminin, gamma 1 (formerly LAMB2) 0.65 FMOD fibromodulin 0.65 MUC4 mucin4, cell surface associated 0.64 EMID1 EMI domain containing 1 0.62 HMCN1hemicentin 1 0.61 NID1 nidogen 1 0.60 VCAN versican 0.58 CILP2 cartilageintermediate layer protein 2 0.57 SOD3 superoxide dismutase 3,extracellular 0.56 ADAMTS3 ADAM metallopeptidase with 0.54thrombospondin type 1 motif, 3 ZP3 zona pellucida glycoprotein 3 0.54(sperm receptor) ANGPTL4 angiopoietin-like 4 0.53 CRTAC1 cartilageacidic protein 1 0.52 LTBP4 latent transforming growth factor 0.50 betabinding protein 4 FREM1 FRAS1 related extracellular matrix 1 0.50Table 6 below lists the extracellular matrix genes that correlate withIsl1 expression with a Pearson's correlation of 0.49-0.00.

TABLE 6 Extracellular matrix genes expressed in HVPs with a PearsonCorrelation with Isl1 Expression of 0.49 to 0.00 Pearson's Correlationwith Gene Extracellular matrix genes (GO: 0031012) Isl1 Expression SSC5Dscavenger receptor cysteine rich family, 5 domains 0.49 GPC6 glypican 60.49 COL1A1 collagen, type I, alpha 1 0.49 ADAMTSL3 ADAMTS-like 3 0.48FLRT3 fibronectin leucine rich transmembrane protein 3 0.48 FBLN1fibulin 1 0.48 ADAMTS9 ADAM metallopeptidase with thrombospondin type 10.48 motif, 9 COL27A1 collagen, type XXVII, alpha 1 0.47 RELN reelin0.46 COL9A2 collagen, type IX, alpha 2 0.46 EFEMP2 EGF containingfibulin-like extracellular matrix protein 2 0.45 AGRN agrin 0.44 PCOLCEprocollagen C-endopeptidase enhancer 0.44 NTN4 netrin 4 0.44 CD248 CD248molecule, endosialin 0.44 TGFB1 transforming growth factor, beta 1 0.43ADAMTS2 ADAM metallopeptidase with thrombospondin type 1 0.43 motif, 2CTHRC1 collagen triple helix repeat containing 1 0.42 FGFR2 fibroblastgrowth factor receptor 2 0.41 APOE apolipoprotein E 0.41 MMP11 matrixmetallopeptidase 11 0.41 MMP15 matrix metallopeptidase 15(membrane-inserted) 0.41 PODN podocan 0.39 VEGFA vascular endothelialgrowth factor A 0.39 COL18A1 collagen, type XVIII, alpha 1 0.39 GLG1golgi glycoprotein 1 0.39 GPC2 glypican 2 0.37 DAG1 dystroglycan 1(dystrophin-associated glycoprotein 1) 0.35 TGFB2 transforming growthfactor, beta 2 0.35 PRELP proline/arginine-rich end leucine-rich repeatprotein 0.35 CHAD chondroadherin 0.33 COL2A1 collagen, type II, alpha 10.33 FN1 fibronectin 1 0.31 SMC3 structural maintenance of chromosomes 30.31 COL4A5 collagen, type IV, alpha 5 0.30 FBN3 fibrillin 3 0.30 MMP23Bmatrix metallopeptidase 23B 0.30 CCDC80 coiled-coil domain containing 800.29 SPARC secreted protein, acidic, cysteine-rich (osteonectin) 0.29TNXB tenascin XB 0.28 COL6A2 collagen, type VI, alpha 2 0.28 ADAMTS13ADAM metallopeptidase with thrombospondin type 1 0.28 motif, 13 LOXL1lysyl oxidase-like 1 0.28 HAPLN2 hyaluronan and proteoglycan linkprotein 2 0.28 TNC tenascin C 0.28 ENTPD2 ectonucleoside triphosphatediphosphohydrolase 2 0.28 TGFB3 transforming growth factor, beta 3 0.28MFAP4 microfibrillar-associated protein 4 0.27 VWF von Willebrand factor0.27 WNT2 wingless-type MMTV integration site family member 2 0.27 MMRN2multimerin 2 0.27 SPON1 spondin 1, extracellular matrix protein 0.26ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 0.26 motif, 1F2 coagulation factor II (thrombin) 0.26 FLRT2 fibronectin leucine richtransmembrane protein 2 0.25 MMP2 matrix metallopeptidase 2 0.24 COL26A1collagen, type XXVI, alpha 1 0.24 CASK calcium/calmodulin-dependentserine protein kinase 0.24 (MAGUK family) NTN3 netrin 3 0.23 SLC1A3solute carrier family 1 (glial high affinity glutamate 0.22transporter), member 3 F3 coagulation factor III (thromboplastin, tissuefactor) 0.20 ADAMTS6 ADAM metallopeptidase with thrombospondin type 10.20 motif, 6 COL5A2 collagen, type V, alpha 2 0.19 ERBB2IP erbb2interacting protein 0.18 LAMB1 laminin, beta 1 0.18 COLQ collagen-liketail subunit (single strand of homotrimer) of 0.18 asymmetricacetylcholinesterase LAMA5 laminin, alpha 5 0.17 LOXL2 lysyloxidase-like 2 0.17 WNT11 wingless-type MMTV integration site family,member 11 0.17 LAMB2 laminin, beta 2 (laminin S) 0.17 COL5A1 collagen,type V, alpha 1 0.17 AEBP1 AE binding protein 1 0.17 COL9A3 collagen,type IX, alpha 3 0.16 CTSD cathepsin D 0.16 COL21A1 collagen, type XXI,alpha 1 0.16 EGFLAM EGF-like, fibronectin type III and laminin G domains0.16 FBN2 fibrillin 2 0.15 NAV2 neuron navigator 2 0.15 EMILIN2 elastinmicrofibril interfacer 2 0.14 WNT9B wingless-type MMTV integration sitefamily, member 9B 0.14 NOV nephroblastoma overexpressed 0.14 CHL1 celladhesion molecule L1-like 0.13 DLG1 discs, large homolog 1 (Drosophila)0.11 MFGE8 milk fat globule-EGF factor 8 protein 0.11 TIMP1 TIMPmetallopeptidase inhibitor 1 0.11 CST3 cystatin C 0.10 APLP1 amyloidbeta (A4) precursor-like protein 1 0.10 PRTN3 proteinase 3 0.10 ADAMTS10ADAM metallopeptidase with thrombospondin type 1 0.09 motif, 10 ILKintegrin-linked kinase 0.09 FRAS1 Fraser extracellular matrix complexsubunit 1 0.09 ANXA2P2 annexin A2 pseudogene 2 0.08 SMOC2 SPARC relatedmodular calcium binding 2 0.07 ANXA2 annexin A2 0.06 ODAM odontogenic,ameloblast asssociated 0.06 FREM2 FRAS1 related extracellular matrixprotein 2 0.05 HAPLN3 hyaluronan and proteoglycan link protein 3 0.05GPC3 glypican 3 0.03 LGALS1 lectin, galactoside-binding, soluble, 1 0.02ADAMTS8 ADAM metallopeptidase with thrombospondin type 1 0.02 motif, 8LUM lumican 0.01 HSP90B1 heat shock protein 90 kDa beta (Grp94), member1 0.00 HAPLN4 hyaluronan and proteoglycan link protein 4 0.00 MATN2matrilin 2 0.00

Extracellular matrix genes whose expression negatively correlated withIsl1 expression in the HVPs were also identified. Table 7 below liststhe extracellular matrix genes that negatively correlate with Isl1expression with a Pearson's correlation of −0.50 or less.

TABLE 7 Extracellular matrix genes expressed in HVPs with a PearsonCorrelation with Isl1 Expression of −0.50 or less Pearson's Correlationwith Gene Extracellular matrix genes (GO: 0031012) Isl1 ExpressionFKBP1A FK506 binding protein 1A, 12 kDa −0.51 CLU clusterin −0.52 TFPI2tissue factor pathway inhibitor 2 −0.52 PLSCR1 phospholipid scramblase 1−0.53 FBLN5 fibulin 5 −0.53 VWA1 von Willebrand factor A domain −0.54containing 1 ADAMTS16 ADAM metallopeptidase with −0.55 thrombospondintype 1 motif, 16 MMP25 matrix metallopeptidase 25 −0.55 SFRP2 secretedfrizzled-related protein 2 −0.61 SOD1 superoxide dismutase 1, soluble−0.68Table 8 below lists the extracellular matrix genes that negativelycorrelate with Isl1 expression with a Pearson's correlation of −0.01 to−0.49.

TABLE 8 Extracellular matrix genes expressed in HVPs with a PearsonCorrelation with Isl1 Expression of −0.01 to −0.49 Pearson's Correlationwith Gene Extracellular matrix genes (GO: 0031012) Isl1 Expression PAPLNpapilin, proteoglycan-like sulfated glycoprotein −0.01 SOST sclerostin−0.01 CDON cell adhesion associated, oncogene regulated −0.02 HMCN2hemicentin 2 −0.02 WNT5A wingless-type MMTV integration site family,member 5A −0.02 PCSK6 proprotein convertase subtilisin/kexin type 6−0.02 GSTO1 glutathione S-transferase omega 1 −0.02 LTBP1 latenttransforming growth factor beta binding protein 1 −0.03 KAZALD1Kazal-type serine peptidase inhibitor domain 1 −0.03 LTBP2 latenttransforming growth factor beta binding protein 2 −0.03 SFRP1 secretedfrizzled-related protein 1 −0.04 ADAM11 ADAM metallopeptidase domain 11−0.05 COL6A1 collagen, type VI, alpha 1 −0.05 COL22A1 collagen, typeXXII, alpha 1 −0.05 CYR61 cysteine-rich, angiogenic inducer, 61 −0.05ELN elastin −0.06 COL9A1 collagen, type IX, alpha 1 −0.06 VTNvitronectin −0.06 COL15A1 collagen, type XV, alpha 1 −0.06 COL4A1collagen, type IV, alpha 1 −0.07 ANG angiogenin, ribonuclease, RNase Afamily, 5 −0.07 HSPG2 heparan sulfate proteoglycan 2 −0.09 CRIP2cysteine-rich protein 2 −0.09 CD151 CD151 molecule (Raph blood group)−0.09 THBS1 thrombospondin 1 −0.09 ADAMTS4 ADAM metallopeptidase withthrombospondin type 1 −0.09 motif, 4 CTGF connective tissue growthfactor −0.10 CRISPLD2 cysteine-rich secretory protein LCCL domaincontaining 2 −0.10 BMP7 bone morphogenetic protein 7 −0.11 COL6A3collagen, type VI, alpha 3 −0.11 COL3A1 collagen, type III, alpha 1−0.11 COL14A1 collagen, type XIV, alpha 1 −0.11 MATN3 matrilin 3 −0.11CPZ carboxypeptidase Z −0.11 BMP1 bone morphogenetic protein 1 −0.11WISP1 WNT1 inducible signaling pathway protein 1 −0.12 ADAMTS18 ADAMmetallopeptidase with thrombospondin type 1 −0.12 motif, 18 COL7A1collagen, type VII, alpha 1 −0.12 IGFBP7 insulin-like growth factorbinding protein 7 −0.12 COCH cochlin −0.13 ADAMTS5 ADAM metallopeptidasewith thrombospondin type 1 −0.13 motif, 5 COL11A2 collagen, type XI,alpha 2 −0.13 TGFBI transforming growth factor, beta-induced, 68 kDa−0.14 COL16A1 collagen, type XVI, alpha 1 −0.14 ACHEacetylcholinesterase (Yt blood group) −0.14 THSD4 thrombospondin, typeI, domain containing 4 −0.15 DGCR6 DiGeorge syndrome critical regiongene 6 −0.15 TGFB1I1 transforming growth factor beta 1 inducedtranscript 1 −0.15 ADAMTSL1 ADAMTS-like 1 −0.15 SERPINA1 serpinpeptidase inhibitor, clade A (alpha-1 antiproteinase, −0.16antitrypsin), member 1 MAMDC2 MAM domain containing 2 −0.16 LAMA4laminin, alpha 4 −0.17 LTBP3 latent transforming growth factor betabinding protein 3 −0.17 EGFL7 EGF-like-domain, multiple 7 −0.17 NPNTnephronectin −0.17 SERPINF1 serpin peptidase inhibitor, clade F (alpha-2antiplasmin, −0.18 pigment epithelium derived factor), member 1 ABI3BPABI family, member 3 (NESH) binding protein −0.18 SERPINE2 serpinpeptidase inhibitor, clade E (nexin, plasminogen −0.18 activatorinhibitor type 1), member 2 WNT6 wingless-type MMTV integration sitefamily, member 6 −0.19 TIMP3 TIMP metallopeptidase inhibitor 3 −0.19SNCA synuclein, alpha (non A4 component of amyloid precursor) −0.19 PKMpyruvate kinase, muscle −0.19 FGF9 fibroblast growth factor 9 −0.19 VITvitrin −0.19 WNT1 wingless-type MMTV integration site family, member 1−0.19 LAMC3 laminin, gamma 3 −0.19 MMP14 matrix metallopeptidase 14(membrane-inserted) −0.19 PXDN peroxidasin −0.19 HNRNPM heterogeneousnuclear ribonucleoprotein M −0.19 FBN1 fibrillin 1 −0.20 ASPN asporin−0.20 ADAMTSL5 ADAMTS-like 5 −0.20 SPON2 spondin 2, extracellular matrixprotein −0.20 COL1A2 collagen, type I, alpha 2 −0.20 BGN biglycan −0.20COL4A2 collagen, type IV, alpha 2 −0.20 ADAMTSL4 ADAMTS-like 4 −0.21APOH apolipoprotein H (beta-2-glycoprotein I) −0.21 CHI3L1 chitinase3-like 1 (cartilage glycoprotein-39) −0.21 ADAMTS7 ADAM metallopeptidasewith thrombospondin type 1 −0.22 motif, 7 CALR calreticulin −0.22 MMP9matrix metallopeptidase 9 −0.22 MMP24 matrix metallopeptidase 24(membrane-inserted) −0.22 SPOCK2 sparc/osteonectin, cwcv and kazal-likedomains −0.22 proteoglycan (testican) 2 COL11A1 collagen, type XI, alpha1 −0.23 MMP7 matrix metallopeptidase 7 −0.23 MMP16 matrixmetallopeptidase 16 (membrane-inserted) −0.23 MFAP2microfibrillar-associated protein 2 −0.23 POSTN periostin, osteoblastspecific factor −0.24 COL8A1 collagen, type VIII, alpha 1 −0.24 WNT2Bwingless-type MMTV integration site family, member 2B −0.24 DCN decorin−0.24 EGFL6 EGF-like-domain, multiple 6 −0.24 MMP10 matrixmetallopeptidase 10 −0.24 MGP matrix Gla protein −0.24 ECM1extracellular matrix protein 1 −0.24 SERPINE1 serpin peptidaseinhibitor, clade E (nexin, plasminogen −0.25 activator inhibitor type1), member 1 MMP1 matrix metallopeptidase 1 −0.25 WNT10A wingless-typeMMTV integration site family, member 10A −0.25 B4GALT7 xylosylproteinbeta 1,4-galactosyltransferase, polypeptide 7 −0.25 COL12A1 collagen,type XII, alpha 1 −0.25 LAMA3 laminin, alpha 3 −0.25 LAMA2 laminin,alpha 2 −0.25 LAMB3 laminin, beta 3 −0.25 WNT7B wingless-type MMTVintegration site family, member 7B −0.25 FLRT1 fibronectin leucine richtransmembrane protein 1 −0.25 ADAMTS15 ADAM metallopeptidase withthrombospondin type 1 −0.26 motif, 15 COL8A2 collagen, type VIII, alpha2 −0.26 MFAP1 microfibrillar-associated protein 1 −0.26 TINAGL1tubulointerstitial nephritis antigen-like 1 −0.26 FGF1 fibroblast growthfactor 1 (acidic) −0.27 OLFML2A olfactomedin-like 2A −0.27 CPA6carboxypeptidase A6 −0.27 COL17A1 collagen, type XVII, alpha 1 −0.27SPARCL1 SPARC-like 1 (hevin) −0.27 MFAP5 microfibrillar associatedprotein 5 −0.27 COL4A4 collagen, type IV, alpha 4 −0.28 WNT8Bwingless-type MMTV integration site family, member 8B −0.28 ADAMTS19ADAM metallopeptidase with thrombospondin type 1 −0.29 motif, 19 CRTAPcartilage associated protein −0.29 WNT5B wingless-type MMTV integrationsite family, member 5B −0.30 WNT3 wingless-type MMTV integration sitefamily, member 3 −0.30 UCMA upper zone of growth plate and cartilagematrix associated −0.30 GPC1 glypican 1 −0.30 TIMP2 TIMPmetallopeptidase inhibitor 2 −0.30 ALPL alkaline phosphatase,liver/bone/kidney −0.30 LECT1 leukocyte cell derived chemotaxin 1 −0.32GPC4 glypican 4 −0.32 SPOCK1 sparc/osteonectin, cwcv and kazal-likedomains −0.32 proteoglycan (testican) 1 HSD17B12 hydroxysteroid(17-beta) dehydrogenase 12 −0.32 LGALS3 lectin, galactoside-binding,soluble, 3 −0.33 EMILIN3 elastin microfibril interfacer 3 −0.34 GFOD2glucose-fructose oxidoreductase domain containing 2 −0.34 VWC2 vonWillebrand factor C domain containing 2 −0.34 SERAC1 serine active sitecontaining 1 −0.34 WNT8A wingless-type MMTV integration site family,member 8A −0.34 LMCD1 LIM and cysteine-rich domains 1 −0.34 CPXM2carboxypeptidase X (M14 family), member 2 −0.34 ADAMTS14 ADAMmetallopeptidase with thrombospondin type 1 −0.34 motif, 14 GPLD1glycosylphosphatidylinositol specific phospholipase D1 −0.35 FGFBP3fibroblast growth factor binding protein 3 −0.35 BCAN brevican −0.35ITGB4 integrin, beta 4 −0.35 LGALS3BP lectin, galactoside-binding,soluble, 3 binding protein −0.36 LPL lipoprotein lipase −0.38 LAD1ladinin 1 −0.39 WNT3A wingless-type MMTV integration site family, member3A −0.39 TGFBR3 transforming growth factor, beta receptor III −0.39 DSTdystonin −0.40 WNT10B wingless-type MMTV integration site family, member10B −0.40 LEFTY2 left-right determination factor 2 −0.41 TNFRSF11B tumornecrosis factor receptor superfamily, member 11b −0.41 WNT9Awingless-type MMTV integration site family, member 9A −0.41 TIMP4 TIMPmetallopeptidase inhibitor 4 −0.42 WNT4 wingless-type MMTV integrationsite family, member 4 −0.42 NCAN neurocan −0.42 ADAMTS20 ADAMmetallopeptidase with thrombospondin type 1 −0.43 motif, 20 ITGA6integrin, alpha 6 −0.43 LOX lysyl oxidase −0.43 THBS4 thrombospondin 4−0.43 THBS2 thrombospondin 2 −0.44 ADAMTSL2 ADAMTS-like 2 −0.44 ENTPD1ectonucleoside triphosphate diphosphohydrolase 1 −0.45 RUNX1runt-related transcription factor 1 −0.45 VWA2 von Willebrand factor Adomain containing 2 −0.45 RELL2 RELT-like 2 −0.46 PTPRZ1 proteintyrosine phosphatase, receptor-type, Z polypeptide 1 −0.46 LAMC2laminin, gamma 2 −0.46 DST dystonin −0.40 WNT10B wingless-type MMTVintegration site family, member 10B −0.40 LEFTY2 left-rightdetermination factor 2 −0.41 TNFRSF11B tumor necrosis factor receptorsuperfamily, member 11b −0.41 WNT9A wingless-type MMTV integration sitefamily, member 9A −0.41 TIMP4 TIMP metallopeptidase inhibitor 4 −0.42

Example 13: Gene Expression Profile for Day 6 Islet 1 Negative Cells

In this example, the gene expression profile was determined for Islet 1negative cells within the Day 6 HVP population to further characterize asubpopulation of cells within the Day 6 population that do not expressthe necessary markers to qualify as engraftable HVPs. Day 6 HVPpopulations were generated as described in Examples 1 or 10 and RNAsequencing (RNA-seq) was performed following differentiation asdescribed in Example 2. Cells that were Islet 1 negative (Isl1−) werefurther analyzed with respect to their gene expression profile. Genesexpressed in the Isl1− cells with an average RNA copy number of 2000 orhigher are shown below in Table 9.

TABLE 9 Gene Expression Profile of Day 6 Islet 1 Negative Cells GeneSample #1 Sample #2 Avg. RNA Copy # ACTB 12288 28126 20207 MTRNR2L224511 9774 17142.5 MALAT1 14163 18092 16127.5 EEF1A1 11663 12456 12059.5KRT8 8884 14087 11485.5 MTRNR2L8 12836 7688 10262 KRT18 5215 105527883.5 FN1 4900 10581 7740.5 MTRNR2L1 8550 5719 7134.5 TTN 3149 96016375 GAPDH 4907 6391 5649 YWHAZ 5349 5414 5381.5 MTRNR2L9 5673 38504761.5 RPL3 3346 5911 4628.5 AHNAK 6727 2197 4462 KCNQ1OT1 5835 30644449.5 TUBB 4870 3936 4403 SLC2A3 3311 4301 3806 FTL 3484 4021 3752.5HSP90B1 4173 2778 3475.5 KRT19 3502 3202 3352 HSPA8 3455 2903 3179 MYL61898 4375 3136.5 RPLP0 2319 3922 3120.5 BSG 2519 3593 3056 COL3A1 5312695 3003.5 TPM1 2938 3059 2998.5 VCAN 2563 3422 2992.5 ENO1 2449 35352992 RPL4 2619 3328 2973.5 ACTG1 2687 3253 2970 MTRNR2L10 3409 2487 2948HMGN2 2684 3153 2918.5 PRTG 2594 2980 2787 TPI1 2418 3113 2765.5 HMGB12577 2880 2728.5 VIM 2621 2704 2662.5 ATP5B 3000 2219 2609.5 HSP90AB12735 2419 2577 RPL7 2132 2896 2514 CBX5 2799 2219 2509 MYL7 1614 33822498 SERPINH1 2547 2327 2437 HNRNPK 2878 1932 2405 SRRM2 2758 2046 2402PODXL 3683 1112 2397.5 EEF2 2579 2119 2349 SPARC 3026 1645 2335.5 ACTC1437 4152 2294.5 HUWE1 2583 1977 2280 COL1A2 3544 941 2242.5 LINC005062965 1496 2230.5 HSPA5 2078 2356 2217 MDK 2223 2144 2183.5 HNRNPC 22922074 2183 HSP90AA1 2220 2138 2179 RGS5 2180 2150 2165 LAMC1 2757 15652161 APLNR 868 3246 2057 UGDH-AS1 2633 1457 2045 RPS3A 1601 2399 2000

Accordingly, the data shown in Table 9 provides a gene expressionprofile for Islet 1 negative, non-engraftable cells within a Day 6 HVPpopulation that are not suitable for transplantation and thus are to beselected against when choosing cells for transplantation andengraftment.

Example 14 Single Cell Sequencing of HVPs

In this example, single cell sequencing was performed on HVPs from day4, 5, 6, 8, 9 or 15 of differentiation to thereby identify expressedgenes in individual cells at different stages of differentiation.Sequencing cell populations in bulk can mask cellular subtypes withinthat population. Single cell sequencing offers an unbiased approach toclassify cell types within a population.

The single cell sequencing approach used herein comprised the threesteps: library preparation, sequencing and analysis. For librarypreparation, a single cell was collected and lysed. The RNAtranscriptome was captured, converted to DNA and prepped for sequencing.The captured transcriptome of a cell is termed a “library”. Sequencingwas performed using a next-generation sequencer. For analysis, thesequences were read and mapped to genes. This created atranscriptome-wide expression profile for each cell. The expressionprofiles were then compared with each other. Similar cells were“clustered” together to represent a cell type.

To obtain the single cells used for single cell sequencing, the HVPdifferentiation was carried out as described previously (see Examples 1and 10). Cells from Day 4, 5, 6, 8, 9 and 15 were collected followingtrypsin treatment, and picked manually with a mouth pipette. 128 cellswere picked for each day. Library preparation for the single cells wasperformed using the Smart-seq2 protocol as previously described(Picelli, S. et al. (2013) Nature Methods 10:1096-1098). The single celllibraries were sequenced on an Illumina HiSeq 2500 Sequencing System.Reads were mapped to the human genome using BowTie (Langmead, B. et al.(2009) Genome Biol. 10(3):R25). Data analysis was carried out using theSeurat package (Sajita, R. et al. (2015) Nature Biotechnology33:495-502).

Using t-SNE dimensionality reduction to visualise the data across Day 4,5, 6, 8, 9 and 15, four major cell types were observed: a non-cardiacpopulation, an epithelial-like population, cardiac progenitors andcardiomyocytes. Differentially expressed genes of the cardiacprogenitors were extracted using the FindMarker function in the Seuratpackage. The parameters were the differentially expressed genes in thecardiac progenitor clusters that were expressed in at least 10% of thecells within the cardiac progenitor cluster. The list of differentiallyexpressed genes in the cardiac progenitor cluster are shown below inTable 10.

TABLE 10 Differentially Expressed Genes in Cardiac Progenitor Clusterp_val avg_diff TMEM88 3.42E−82 1.560606117 COL3A1 3.79E−80 1.550451213MIR3606 3.79E−80 1.550451213 APLNR 1.80E−51 1.342551564 HAS2 7.87E−551.302299387 MFAP4 1.89E−103 1.287366398 LIX1 2.20E−81 1.262443162 LUM2.06E−48 1.121559334 PCOLCE 6.13E−80 1.096776495 HAND1 2.20E−311.090769383 HEY1 2.64E−56 1.052143244 PDGFRA 4.21E−102 1.029400192S100A10 2.82E−41 0.99881898 IGF2 2.44E−62 0.945955427 MMP2 1.43E−710.910583615 COL1A1 9.55E−58 0.894758619 ART5 1.16E−23 0.883791494 LRRN43.99E−36 0.84972281 H19 2.73E−35 0.8408468 MIR675 2.73E−35 0.8408468INS.IGF2 6.12E−46 0.756272517 S100A11 9.17E−53 0.745858163 SHISA33.45E−12 0.73714255 RGS2 8.20E−18 0.731496568 SLC9A3R1 4.75E−260.723600997 MIR3615 4.75E−26 0.723600997 ITM2C 1.77E−48 0.720322592CYP27A1 2.14E−26 0.700002885 BMPER 3.56E−42 0.696442368 SPARC 4.07E−410.691895813 NID2 1.35E−39 0.688341218 PCOLCE.AS1 2.29E−72 0.684407943PLAT 1.46E−69 0.677075789 RGS5 1.66E−41 0.672985639 RGS4 4.78E−310.671536299 FBLN1 3.10E−46 0.664157944 GATA6.AS1 3.55E−24 0.659546503EIF4EBP1 1.29E−36 0.656965269 HAPLN1 2.53E−20 0.65191049 COL1A2 1.99E−290.64867676 CDH3 5.74E−34 0.64562218 NXPH2 5.44E−23 0.630747775 BST22.63E−10 0.630640659 APOBEC3C 1.43E−38 0.627430539 SFRP5 2.01E−390.625478497 KRT19 3.22E−37 0.625378553 HOXB2 8.19E−33 0.602689448 CHIC26.53E−30 0.60132243 BMP5 5.89E−31 0.599081591 CALB2 6.82E−16 0.596591168CFC1 1.44E−54 0.594202643 KRT18 2.71E−36 0.590624042 LRRTM1 9.36E−430.584576973 GYPC 1.22E−47 0.58078756 MEIS1 1.71E−57 0.58057826 KRT82.54E−43 0.577311366 TUBB6 2.02E−21 0.571891695 PBX3 3.17E−390.570615432 TNC 6.89E−36 0.570340016 CTSV 3.09E−17 0.56938514 ALX12.64E−14 0.552977549 FOXH1 5.22E−26 0.548734193 VIM.AS1 4.78E−280.542791752 TMEM141 6.48E−27 0.538575158 GATA6 1.63E−46 0.537437948 BMP48.51E−15 0.531659308 VIM 2.40E−29 0.530724516 ARL4D 2.48E−16 0.51292713HBE1 2.21E−14 0.505457358 CD248 3.74E−19 0.503966584 GLIPR2 9.74E−300.502526018 TMEM185A 6.17E−39 0.49852561 KIAA1462 7.15E−75 0.497738542FSHR 5.25E−45 0.497054658 HTRA1 3.27E−13 0.495987153 SPP1 8.45E−090.494008412 IGFBP4 7.48E−14 0.490252699 NRP1 8.31E−43 0.484466262 SC5D1.02E−22 0.482101626 FAM89A 5.27E−26 0.477661385 MIR1182 5.27E−260.477661385 LOC400043 3.49E−18 0.475045692 ASH2L 1.14E−46 0.474197679PKP2 1.44E−25 0.474140269 FN1 5.68E−18 0.469988478 KDR 8.18E−300.463258104 BGN 2.90E−23 0.459541322 CREG1 7.00E−13 0.459398822 PTGIS7.28E−39 0.456120126 DSP 6.66E−24 0.454180797 PKIG 5.19E−24 0.452582156CTSB 7.02E−23 0.449928855 KLHL4 2.64E−40 0.449780298 CCDC34 2.05E−330.443083183 ATF7IP2 1.86E−59 0.440835688 KIAA1551 3.78E−27 0.439543958SLC40A1 1.11E−23 0.439358025 SDC4 8.23E−17 0.437190914 IGFBP3 6.90E−070.436357742 SERINC3 1.64E−23 0.425115033 GUCY1A3 7.48E−28 0.424214515CCDC3 1.41E−35 0.422569528 TGFBI 6.52E−16 0.41635348 LAMP5 8.54E−060.415693362 COL6A1 2.88E−25 0.407932515 PLS3 8.22E−12 0.406911474LAPTM4A 1.16E−25 0.405037615 RUNX1T1 7.93E−78 0.403683684 FGF10 2.18E−130.401291848 LRIG3 1.89E−30 0.38790471 DSG2 6.23E−28 0.387712513 ARL2BP7.58E−14 0.38444917 TCEAL9 1.70E−21 0.38384913 PAPSS1 1.40E−140.378653665 ANXA6 2.68E−21 0.378214393 LGALS1 2.64E−06 0.378105889 FSTL12.78E−24 0.376247551 MIR198 2.78E−24 0.376247551 TMEM98 8.37E−190.376170536 SARAF 4.16E−19 0.375950055 TP53I11 6.05E−17 0.373187071CYB5D1 1.63E−23 0.372197782 IFITM3 7.56E−38 0.371288978 LGR5 1.34E−160.369873765 WASF1 9.73E−17 0.368716853 ECE1 9.71E−35 0.364724949 MXRA82.10E−12 0.363862558 CSRP2 2.60E−17 0.362236698 SDCBP 6.33E−190.361772835 SLC16A1 9.31E−11 0.355359606 COL6A2 1.73E−24 0.353551929EMP2 3.26E−30 0.352860372 LEPROTL1 2.29E−27 0.352310781 CA2 5.04E−080.349789326 CCBE1 9.52E−35 0.349748013 SMOC2 1.94E−17 0.347190203 HOXB12.81E−17 0.346717419 GATA5 7.66E−10 0.344901285 RBP1 8.58E−21 0.34419898PHLDA1 3.77E−28 0.343778762 HAND2 2.82E−24 0.343032467 SLC30A3 2.09E−210.335148941 SERPINE2 5.79E−22 0.334593289 SYT10 1.62E−39 0.332905271RHOC 1.16E−16 0.332380866 MPZL1 1.54E−20 0.330849688 LAPTM4B 1.72E−110.330782753 LRRC32 1.75E−22 0.327573069 CGNL1 4.43E−31 0.32754955 CXCL121.71E−14 0.326028623 NODAL 7.70E−06 0.324782701 DOK4 5.00E−100.322776109 ALDH2 1.15E−24 0.322496612 LOC101927497 5.25E−23 0.321216676RAMP2 3.25E−12 0.320975405 PLSCR5 5.92E−24 0.320022623 COL5A2 4.08E−280.319401842 FAM114A1 7.04E−28 0.319200887 METTL7A 1.05E−53 0.319011726CYYR1 1.65E−18 0.318957318 TOMM34 1.96E−07 0.318041963 CDH11 2.13E−210.317968138 FLRT3 8.21E−08 0.316560332 LYPD6 9.56E−23 0.316391668AFAP1L2 5.04E−32 0.315613744 SERPINB9 5.60E−12 0.315004476 DYNC1I11.11E−20 0.313338566 MGARP 1.62E−09 0.311406096 PHGDH 1.17E−090.310776152 FHL3 8.79E−08 0.309585103 NPY 2.27E−05 0.309536196 PPP1R15A2.34E−09 0.309484964 SPIN4 4.33E−24 0.309135856 FXYD6.FXYD2 5.50E−100.308833983 P3H2 9.86E−16 0.307905833 FMOD 1.98E−12 0.307428412 FAM213A8.18E−22 0.305776171 DHDH 3.35E−08 0.303929476 ARSE 3.34E−11 0.302502476BRINP1 2.18E−16 0.300841535 MYH10 6.61E−15 0.299656764 HSPA5 2.65E−110.29901021 ARF4 1.65E−12 0.297788482 CD74 2.28E−11 0.296915796 WNT5A2.27E−44 0.29686904 IL6ST 1.62E−40 0.296859117 B2M 6.62E−25 0.296077624MDK 1.00E−32 0.295677643 TIMP1 4.82E−11 0.294947926 PDIA4 1.34E−080.294630611 NPPB 0.012639596 0.29392058 RAB34 8.90E−13 0.293594025TAX1BP3 2.28E−06 0.293246644 SVEP1 1.02E−62 0.291659157 CADM1 4.45E−190.29143391 EMP3 2.86E−12 0.291231978 ISL1 9.26E−08 0.289460012 CSNK2A21.33E−21 0.288992825 SERPING1 6.85E−12 0.287793225 TECRL 4.21E−050.286902439 SLC7A2 2.65E−37 0.286569819 GPR108 1.88E−10 0.284415977MIR6791 1.88E−10 0.284415977 KDM6A 4.24E−13 0.283535404 RDH10 6.97E−330.283087084 PLOD1 3.49E−09 0.280933842 COPG1 5.98E−11 0.279866228 CASP66.03E−09 0.279773211 THBS1 8.65E−06 0.279660845 H2AFY2 1.19E−060.278216329 GOLGA7 1.10E−10 0.276228148 SELENOF 7.41E−11 0.274011316FXYD6 3.31E−10 0.273563514 POSTN 2.13E−28 0.273267932 EFEMP2 3.05E−200.273241569 TGFB2 2.05E−13 0.273044307 TPM4 1.38E−14 0.272429895 RAB380.000686908 0.271918483 NCCRP1 1.79E−11 0.271623914 SH3GLB1 1.51E−180.269224524 PMP22 1.57E−07 0.268878282 PLLP 4.96E−35 0.268507068 SCMH12.01E−09 0.267191985 MIR503HG 1.86E−07 0.265375187 MIR424 1.86E−070.265375187 KDELR2 9.12E−10 0.264599726 DUSP6 0.000293032 0.263830821SERPINH1 3.02E−17 0.263654992 PTGES 2.95E−05 0.263223829 TMEM120A4.23E−07 0.263152389 ADAM19 5.67E−22 0.261893827 CRB2 1.47E−120.261335033 ADA 2.80E−10 0.260801645 PGF 3.62E−22 0.259993454 NCAM11.73E−28 0.259741889 NKX3.1 1.80E−15 0.259560142 COLEC11 1.56E−260.259066004 LOC101927230 2.52E−09 0.257688783 RCN3 2.90E−06 0.256437487ADAMTS12 1.28E−25 0.255820711 SNORD36B 0.01966799 0.255563423 HOXB.AS32.31E−58 0.254873973 STAR 9.39E−29 0.254111765 TMED2 8.28E−190.253051723 CAPG 2.11E−08 0.252316312 APOC1 1.79E−08 0.25228836 FKBP76.16E−14 0.25196672 PTPN13 2.10E−14 0.250842517 EHD2 4.27E−140.250458307 C1orf168 2.14E−15 0.250376891

From the list of genes, neuropilin 1 (NRP1) was selected as a candidatefor positive sorting of HVPs. An anti-NRP1 antibody compatible for MACSsorting is commercially available.

The day 5 dataset was further examined, since RNA expressed on day 5would be translated into protein on day 6, the key window where thesecells could engraft. The tSNE plot of the day 5 dataset, shown in FIG.11, reveals two clusters of cells with similar expression patterns,arbitrarily labeled 0 and 1. Cells within a cluster express similargenes. Cells in different clusters express different genes. The distancebetween each cell on the tSNE plot represents the degree of differencein their gene expression profile. The cluster labeled 1 has high levelsof Isl1 expression.

FIG. 12 shows the expression of Isl1 and NRP1 on day 5 cells, whereindark grey denotes high expression, middle grey denotes low expressionand light grey denotes no expression. Thus, FIG. 12 demonstrates thatmany of the cells with high levels of Isll expression also have highlevels of NRP1 expression.

This was confirmed by the violin plot of expression levels of Isl1 andNRP1 shown in FIG. 13. Cells within cluster 1 (shown in FIG. 11) havehigh levels of expression of Isl1. The violin plot of FIG. 13demonstrates that Isl1+ cells and NRP1+ cells are in the same cluster,since they have a similar gene expression profile. Accordingly, NRP1 isa suitable cell surface marker for positive selection of HVPs (e.g., day5-6 Isl1+ HVPs).

Example 15 Further Characterization of NRP1+ HVPs

In this example, the expression of NRP1 on a human embryonic stem cellline was examined further. The H9 stem cell line (also known in the artas WA09; Thomson, J. A. et al. (1998) Science 282:1145-1147; WiCellResearch Institute) was used for these experiments. H9 cells werecultured under cardiac differentiation conditions (e.g., as describedherein in Examples 1 and 10) to generate day 6 human ventricularprogenitor cells (HVPs).

In a first set of experiments, day 6 HVPs from H9 cells were stainedwith anti-NRP1, anti-TRA-1-60 or both anti-NRP1 and anti-TRA-160,followed by flow cytometry analysis. The results are shown in FIG. 14A(single staining for NRP1+ cells), FIG. 14B (single staining forTRA-1-60+ cells) and FIG. 14C (double staining for NRP1+ TRA-1-60+cells). The results determined that 92% of the day 6 HVPs were NRP1+ and17% of the day 6 HVPs were TRA-1-60+. The double staining analysisdetermined that within the NRP1+ population, 15.5% of those cells areTRA-1-60+.

In a second set of experiments, day 6 HVPs from H9 cells were stainedwith anti-NRP1, anti-ISL1 or both anti-NRP1 and anti-ISL1, followed byflow cytometry analysis. The results are shown in FIG. 15A (singlestaining for NRP1+ cells), FIG. 15B (single staining for ISL1+ cells)and FIG. 15C (double staining for NRP1+ ISL1+ cells). The resultsdetermined that 78% of the day 6 HVPs were NRP1+, 80% of the day 6 HVPswere ISL1+ and 73% of the day 6 HVPs were NRP1+ ISL1+.

These results confirm the expression of NRP1 on the surface of a largemajority of the day 6 HVPs, as well as coexpression with ISL1 and withTRA-1-60, thereby confirming the suitability of NRP1 as a positiveselection, ISL1 as a positive co-selection marker, and TRA-1-60 as anegative selection marker.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for isolating human cardiac ventricular progenitor cells,the method comprising: contacting a culture of human cells containingcardiac progenitor cells with one or more agents reactive withneuropilin-1 (NRP1); and separating NRP1 reactive positive cells fromnon-reactive cells to thereby isolate human cardiac ventricularprogenitor cells.
 2. The method of claim 1, wherein the culture of humancells is further contacted with at least one second agent reactive withhuman cardiac ventricular progenitor cells; and NRP1 reactive/secondagent reactive positive cells are separated from non-reactive cells tothereby isolate human cardiac ventricular progenitor cells.
 3. Themethod of claim 2, wherein the at least one second agent is reactivewith JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9.
 4. The method of claim 2,wherein the culture of human cells is contacted with the agent reactivewith NRP1 before contacting with the at least one second agent.
 5. Themethod of claim 2, wherein the culture of human cells is contacted withthe at least one second agent before contacting with the agent reactivewith NRP1.
 6. The method of claim 2, wherein the culture of human cellsis contacted simultaneously with the agent reactive with NRP1 and withthe at least one second agent.
 7. The method of claim 1, which furthercomprises contacting the human cardiac ventricular progenitor cells withone or more agents reactive with at least one marker that is expressedon the surface of human pluripotent stem cells and separatingmarker-nonreactive negative cells from reactive cells to thereby furtherisolate human cardiac ventricular progenitor cells.
 8. The method ofclaim 7, wherein the at least one marker that is expressed on thesurface of human pluripotent stem cells is selected from the groupconsisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9,CD24, E-cadherin and Podocalyxin, and combinations thereof.
 9. Themethod of claim 7, wherein the at least one marker that is expressed onthe surface of human pluripotent stem cells is TRA-1-60.
 10. The methodof claim 9, wherein the agent reactive with TRA-1-60 is an anti-TRA-1-60antibody.
 11. The method of claim 1, wherein the agent reactive withNRP1 is an anti-NRP1 antibody.
 12. The method of claim 1, wherein theagent reactive with NRP1 is a soluble NRP1 ligand fusion protein. 13.The method of claim 12, wherein the NRP1 ligand is VEGF-A or Sema3A. 14.The method of claim 1, wherein the NRP1 reactive positive cells areseparated from the non-reactive cells by fluorescent activated cellsorting (FACS) or magnetic activated cell sorting (MACS).
 15. The methodof claim 1, wherein the human cardiac ventricular progenitor cells arefurther cultured and differentiated such that they are MLC2v positive.16. The method of claim 1, wherein the culture of human cells containingcardiac progenitor cells is derived from human embryonic stem cells ofhuman induced pluripotent stem cells.
 17. A method for isolating humancardiac ventricular progenitor cells, the method comprising: culturinghuman pluripotent stem cells under conditions that generate cardiacprogenitor cells to obtain a culture of cells; contacting the culture ofcells with one or more agents reactive with neuropilin-1 (NRP1); andseparating NRP1 reactive positive cells from non-reactive cells tothereby isolate human cardiac ventricular progenitor cells.
 18. Themethod of claim 17, wherein the culture of human cells is furthercontacted with at least one second agent reactive with human cardiacventricular progenitor cells; and NRP1 reactive/second agent reactivepositive cells are separated from non-reactive cells to thereby isolatehuman cardiac ventricular progenitor cells.
 19. The method of claim 18,wherein the at least one second agent is reactive with JAG1, FZD4, LIFR,FGFR3 and/or TNFSF9.
 20. The method of claim 18, wherein the culture ofhuman cells is contacted with the agent reactive with NRP1 beforecontacting with the at least one second agent.
 21. The method of claim18, wherein the culture of human cells is contacted with the at leastone second agent before contacting with the agent reactive with NRP1.22. The method of claim 18, wherein the culture of human cells iscontacted simultaneously with the agent reactive with NRP1 and with theat least one second agent.
 23. The method of claim 17, which furthercomprises contacting the human cardiac ventricular progenitor cells withone or more agents reactive with at least one marker that is expressedon the surface of human pluripotent stem cells and separatingmarker-nonreactive negative cells from reactive cells to thereby furtherisolate human cardiac ventricular progenitor cells.
 24. The method ofclaim 23, wherein the at least one marker that is expressed on thesurface of human pluripotent stem cells is selected from the groupconsisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9,CD24, E-cadherin and Podocalyxin, and combinations thereof.
 25. Themethod of claim 23, wherein the at least one marker that is expressed onthe surface of human pluripotent stem cells is TRA-1-60.
 26. The methodof claim 25, wherein the agent reactive with TRA-1-60 is ananti-TRA-1-60 antibody.
 27. The method of claim 17, wherein the agentreactive with NRP1 is an anti-NRP1 antibody.
 28. The method of claim 17,wherein the agent reactive with NRP1 is a soluble NRP1 ligand fusionprotein.
 29. The method of claim 28, wherein the NRP1 ligand is VEGF-Aor Sema3A.
 30. The method of claim 17, wherein the NRP1 reactivepositive cells are separated from the non-reactive cells by fluorescentactivated cell sorting (FACS) or magnetic activated cell sorting (MACS).31. The method of claim 17, wherein the human cardiac ventricularprogenitor cells are further cultured and differentiated such that theyare MLC2v positive.
 32. The method of claim 17, wherein the culture ofhuman cells containing cardiac progenitor cells is derived from humanembryonic stem cells of human induced pluripotent stem cells.
 33. Amethod for obtaining a clonal population of human cardiac ventricularprogenitor cells, the method comprising: isolating a single NRP1+ humancardiac ventricular progenitor cell by contacting a culture of humancardiac ventricular progenitor cells with one or more agents reactivewith NRP1; and culturing the single NRP1+ human cardiac ventricularprogenitor cell under conditions such that the cell is expanded to atleast 1×10⁹ cells to thereby obtain a clonal population of human cardiacventricular progenitor cells.
 34. The method of claim 33, wherein thesingle NRP1+ human cardiac ventricular progenitor cell is Islet 1positive, Nkx2.5 negative and flk1 negative at the time of initialculture.
 35. The method of claim 33, wherein the single NRP1+ humancardiac ventricular progenitor cell is isolated by fluorescent activatedcell sorting or magnetic activated cell sorting.
 36. The method of claim33, wherein the agent reactive with NRP1 is an anti-NRP1 antibody. 37.The method of claim 33, wherein the single NRP1+ human cardiacventricular progenitor cell is cultured under conditions such that thecell is biased toward ventricular differentiation.
 38. The method ofclaim 33, wherein the single NRP1+ human cardiac ventricular progenitorcell is expanded to at least 10×10⁹ cells.
 39. A clonal population of atleast 1×10⁹NRP1+ human cardiac ventricular progenitor cells obtained bythe method of claim
 33. 40. A clonal population of at least 10×10⁹NRP1+human cardiac ventricular progenitor cells obtained by the method ofclaim
 38. 41. A method of enhancing cardiac function in a subject, themethod comprising administering a pharmaceutical composition comprisingthe clonal population of claim 39 to the subject.
 42. The method ofclaim 41, wherein the clonal population is administered directly intothe heart of the subject.
 43. The method of claim 41, wherein thesubject has suffered a myocardial infarction.
 44. The method of claim41, wherein the subject has a congenital heart disorder.
 45. The methodof claim 42, wherein the clonal population is administered directly intoa ventricular region of the heart of the subject.
 46. The method ofclaim 41, wherein the pharmaceutical composition comprises the clonalpopulation formulated onto a two dimensional or three dimensionalmatrix.
 47. A method for generating human ventricular tissue comprising:transplanting NRP1+ human cardiac ventricular progenitor cells into anorgan of a non-human animal; and allowing the progenitor cells to growin vivo such that human ventricular tissue is generated.
 48. The methodof claim 47, wherein the non-human animal is an immunodeficient mouse.49. The method of claim 47, wherein the organ is a kidney or a heart.50. The method of claim 47, wherein the cells are transplanted at a timewhen one, two, three, four or five of the following cell marker patternsare present: (i) after peak of cardiac mesoderm formation; (ii) at timeof peak Islet-1 expression; (iii) before peak of NKX2.5 expression; (iv)before peak expression of downstream genes MEF-2 and TBX-1; and (v)before expression of differentiated contractile protein genes.
 51. Themethod of claim 47, wherein the cells are transplanted between day 5 andday 7 (inclusive) of in vitro culture of human pluripotent stem cellsunder conditions to generate human ventricular progenitor cells.
 52. Themethod of claim 51, wherein the cells are transplanted on day 6 of invitro culture of human pluripotent stem cells under conditions togenerate human ventricular progenitor cells.
 53. A method of screeningfor cardiac toxicity of test compound, the method comprising: providingNRP1+ human cardiac ventricular progenitor cells; contacting the cellswith the test compound; and measuring toxicity of the test compound forthe cells, wherein toxicity of the test compound for the cells indicatescardiac toxicity of the test compound.